Method and System for Reducing the Likelihood of a Porphyromonas Gingivalis Infection in a Human Being

ABSTRACT

A method and system of reducing the likelihood of a  Porphyromonas gingivalis  infection in a human being includes the use of a bioadhesive strip that binds to a person&#39;s mucosal membrane for at least 1 hour while inside a person&#39;s mouth, where the strip has a therapeutically effective amount of an anti-biofilm agent, compounds that facilitate the growth of desired bacteria beneficial to a person&#39;s health, and an antibiotic effective amount to kill  Porphyromonas gingivalis  bacteria. The method and system reduces the virulence factor gingipain. In other embodiments, the likelihood that a subject will suffer from Alzheimer&#39;s Disease is reduced by administering, via local gingival application to a subject that has been diagnosed with periodontitis, an effective amount of an antibiotic effective to kill  Porphyromonas gingivalis  bacteria residing on the subgingival tooth area of the subject.

RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 16/020,433, filed Jun. 27, 2018 (now U.S. Pat. No.10,583,033, issued Mar. 10, 2020), which is a continuation-in-partapplication of U.S. Ser. No. 15/342,642, filed Nov. 13, 2016 (now U.S.Pat. No. 10,010,568, issued Jul. 3, 2018), which seeks priority fromU.S. Provisional Patent Application Ser. No. 62/260,906, filed Nov. 30,2015.

The present application is a continuation-in-part of U.S. Ser. No.15/270,034, filed Sep. 20, 2016 (now U.S. Pat. No. 9,750,802, issuedSep. 5, 2017), which is a continuation-in-part of U.S. patentapplication Ser. No. 14/954,074, filed Nov. 30, 2015 (now U.S. Pat. No.9,457,077, issued Oct. 4, 2016), which is a continuation-in-part of U.S.patent application Ser. No. 14/574,517, filed Dec. 18, 2014, (now U.S.Pat. No. 9,408,880, issued Aug. 9, 2016), which claims priority fromU.S. Provisional Patent Application Ser. No. 62/072,476, filed on Oct.30, 2014, U.S. Provisional Patent Application Ser. No. 62/053,926, filedSep. 23, 2014, U.S. Provisional Patent Application Ser. No. 62/014,855,filed Jun. 20, 2014 and U.S. Provisional Patent Application Ser. No.61/919,297, filed on Dec. 20, 2013.

The present application also is a continuation-in-part of U.S. patentapplication Ser. No. 15/228,454, filed Aug. 4, 2016 (now U.S. Pat. No.9,585,920, issued Mar. 7, 2017).

This application is also a continuation-in-part of U.S. patentapplication Ser. No. 14/752,192 filed on Jun. 26, 2015 (now U.S. Pat.No. 9,549,842, issued Jan. 24, 2017) which is a continuation-in-part ofU.S. patent application Ser. No. 14/225,503, filed on Mar. 26, 2014,(now U.S. Pat. No. 9,445,936, issued Sep. 20, 2016) which is acontinuation of U.S. patent application Ser. No. 13/367,052, filed onFeb. 6, 2012 (now U.S. Pat. No. 8,701,671, issued on Apr. 22, 2014), andclaims priority from U.S. Provisional Patent Application Ser. No.61/556,023 filed Nov. 4, 2011 and U.S. Provisional Patent ApplicationSer. No. 61/439,652 filed on Feb. 4, 2011. The entire disclosure of theprior applications are considered to be part of the disclosure of theaccompanying application and are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention, in various embodiments, is directed to thetargeting of the microbiome of an individual to promote health and totreat inflammatory diseases, including specifically Alzheimer's disease,by the modification of an individual's oral microbiome and effectivemanagement of oral health care. The cause of the vast majority ofAlzheimer's Disease (AD) is submitted to be periodontitis, and thus, ADis both treatable and preventable by targeting the oral microbiome toprevent later infection of brain tissue by oral spirochetes.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a neurodegenerative disease whichsignificantly increases with age. Currently there is no cure forAlzheimer's disease and ultimately it results in death. It is estimatedthat in 2014, there were 5.2 million Americans living with Alzheimer'sdisease, two thirds consisting of women. One in six women are estimatedto be at risk for developing Alzheimer's. Alzheimer's disease is the 6thleading cause of death in the United States, and approximately 500,000people die each year from the disease. It is the most expensivecondition in the U.S. to treat, with the direct cost to the U.S. ofcaring for those with Alzheimer's exceeding $200 billion, including $150billion in costs to Medicare and Medicaid. It is estimated that thesecosts will reach $1.2 trillion in 2050, as it is expected that 16million Americans will be living with Alzheimer's disease at that time.

Alzheimer's disease was first described by the Bavarian psychiatristAlois Alzheimer in 1907. It is a progressive neuropsychiatric disorderwhich begins with short term memory loss and proceeds to loss ofcognitive functions, disorientation, impairment of judgment andreasoning and, ultimately, dementia. AD is the most common form ofdementia. There is currently no treatment for AD that can reverse orslow down the disease progression. AD represents a major health problemand an effective treatment of the disease would represent a majorbreakthrough.

The clinical manifestations of Alzheimer's disease begin with subtleshort-term memory deficits and depressive symptoms, followed byorientation and language difficulties. Intellectual functionsprogressively disappear and patients become entirely dependent,typically surviving in this devastating state for more than a decade.Death generally occurs from a secondary infection, frequently frompneumonia or urinary infection. The duration of the disease from theappearance of the first symptoms and the manifestation of dementiavaries between 5 and 20 years. Clinical diagnosis of Alzheimer's diseaseoften occurs long after the onset of the disease. It is usually firstnoticed by immediate family members who detect problems with short-termmemory and unusual behavior. Confirmation is achieved post-mortem bydetecting the presence of the pathological hallmarks of the disease,amyloid plaques and neurofibrillary tangles.

The mainstream current FDA-approved pharmacological treatment forAlzheimer's disease is to ameliorate cognitive decline by restoringneurotransmitter signaling between neurons. Four of the five medicationsapproved by the FDA for prescription to Alzheimer's disease patients areaimed at increasing extracellular levels of acetylcholine by delayingits degradation (the acetylcholinesterase inhibitors: tacrine,rivastigmine, galantamine and donepezil). The fifth approved drug ismemantine, a partial antagonist for the ionotropic glutamate NMDAreceptor. Memantine presumably reduces calcium-mediated glutamateexcitotoxicity, hence slowing synaptic and neuronal losscharacteristically observed in the Alzheimer's diseased brain. Althoughthese treatments have proven to maintain cognitive function inAlzheimer's diseased patients, the therapeutic effect is transient andprimarily symptomatic. These treatments are not mechanism-based and donot seem to have any disease-modifying effects. Moreover, no significantproof supports that these drugs play a role in the prevention ofcognitive decline or the reversal of pathological hallmarks andneurodegeneration in Alzheimer's disease.

“The causes of Alzheimer's disease remain unknown and there is no cure.”August 2014 issue of the Joumal of Alzheimer's Disease. Previoussuggestions that particular genetic factors were involved in Alzheimer'sdisease, e.g. the E4 allele, and associations of APOE and the disease,have been shown to be incorrect, as there is no evidence for linkagebetween Alzheimer's disease and APOE.

There is, as yet, no cure for Alzheimer's disease despite concertedefforts and investment by industry. There is therefore a long-felt butunsolved need for a method and system to prevent Alzheimer's disease inorder to spare tens of millions of individuals the terrible fateotherwise awaiting them.

SUMMARY OF THE INVENTION

The present invention provides a method and system for reducing thelikelihood that one will suffer from Alzheimer's disease and thus,preventing Alzheimer's disease in a manner that deals with the causativeagents of the disease. The present invention is focused on steps thatcan be taken decades in advance of the symptoms of Alzheimer's diseasefirst appearing. For the first time, the present invention provides away for individuals to adopt positive behaviors, techniques and usecompositions and products that can thwart the progression of Alzheimer'sdisease. The present invention is directed, from a more generalperspective, on the human microbiome and its relationship toinflammatory responses related to chronic diseases that have, to date,been unresolved and misunderstood. The relationship between the oralmicrobiome and Alzheimer's disease is focused on to determine aneffective method and system to prevent the initiation and theprogression of the disease.

Humans have co-evolved with micro-organisms and have a symbiotic ormutualistic relationship with their resident microbiomes. As at otherbody surfaces, the mouth has a diverse microbiota that grows on oralsurfaces as structurally and functionally organized biofilms. Onoccasions, the oral microbiota breaks down, and previously minorcomponents of the microbiota outcompete beneficial bacteria, therebyincreasing the risk of disease.

There is increasing evidence that the reach of gut and oral microbesextends beyond the intestine, affecting systemic processes, such asmetabolism and organ functions of the brain, the cardiovascular system,liver, and other organs. Several metabolomic studies have identifiedhundreds of compounds in blood that are specifically derived ordependent on the presence of gut microbes.

Researchers have analyzed people's gut bacteria by their occupation andhave found that those who had regular contact with livestock, such asfarmers and their wives, had bacterial communities dominated byPrevotella, a type of bacteria that is also abundant in the gutmicrobiota of cattle and sheep. The anaerobic black-pigmented Prevotellaintermedia is a Gram-negative rod-shaped bacterium whose habitat is thestrictly anaerobic environments of the gastrointestinal tract andgingival crevice. P. intermedia plays an important role in the onset andsubsequent development of polymicrobial periodontal diseases. It isknown that Prevotella intermedia adaptation to oxidative stressinfluences the virulence of the microorganism.

One aspect to the mystery of Alzheimer's disease etiopathogenesis liesin the relative absence of Alzheimer's disease in certain Amishcommunities. The prevalence of Prevotella in the oral cavity of theAmish, the production of nitric oxide by this bacteria, and thereduction of spirochetes in the brains of the Amish, all presentevidence as to an effective avenue for therapeutic intervention inAlzheimer's disease prevention and/or progression. The insights gainedfrom an evaluation of the Amish, especially with respect to the oralmicrobiome they possess and the absence of various diseases they sufferfrom, is one of the foundational aspects of various embodiments of thepresent invention. In some embodiments, mimicking particular conditionsof the Amish is accomplished to achieve some of the benefits derivedfrom the oral microbiome established in certain Amish populations.

One aspect of the present invention is also directed to the pathology ofAlzheimer's disease as it relates to inflammation. Reactive microgliaand astrocytes adjacent to Aβ plaques is a common observation in thebrain of Alzheimer's disease sufferers. It is thought that activatedglia is at first beneficial for degrading Aβ plaques. Chronicinflammation, however, leads to the production of several cytokines thathave been demonstrated to exacerbate other Alzheimer's diseasepathologies. An increased infectious burden and higher serum levels ofinflammatory cytokines have been associated with serum Aβ markers inAlzheimer's disease patients. Chronic infections caused by thesepathogens have been shown to result in cardio-cerebral vasculardisorders, which subsequently promote the development of Alzheimer'sdisease. These studies provide supporting evidence that accumulativeinfections are associated with Alzheimer's disease and supports the roleof infection and inflammation in the etiopathogenesis of Alzheimer'sdisease. Certain aspects of the present invention are directed to areduction in the risk of Alzheimer's disease through a combination ofantibiotic and anti-inflammatory therapy.

The fact that Alzheimer's disease usually develops in later lifesuggested that a slow-acting unconventional infectious agent acquired atan early age and requiring decades to become active may be involved inits etiology. Oral spirochetes are such unconventional infectiousagents. Spirochetes have been found in about 90% of Alzheimer'spatients, while these bacteria were virtually absent in the brains ofhealthy age-matched controls. Once a spirochete infection begins in thebrain, it causes disease by having plaques or masses formed along thecerebral cortex. It is believed that such plaque formation is a normalimmune response to an invasive bacteria in the brain tissue. AβP is themain component of amyloid plaques and is instrumental in thepathogenesis of Alzheimer's disease. In Alzheimer's disease, the brain'snormal defenses become dysfunctional as the macrophages (microglia)become trapped and then attacked within the core of the spirocheteplaque. With immune dysfunction setting in, the spirochete infectionintensifies involving more and more brain cells. Damaged brain cellsproduce amyloid-beta protein as an adaptive response to the infection.As an adaptive response to infectious organisms, like invadingspirochetes, amyloid-beta protein is produced, which we now know hasanti-bacterial properties. Amyloid-beta deposits grow and begin toaffect brain cell connections and communication highways. With damagedconnections and communication highways, dementia symptoms begin andgradually worsen.

Spirochetes are a commonly isolated microorganism in moderate to severeperiodontitis, suggesting that periodontopathic bacteria can invade thebrain by systemic circulation as well as via peripheral nerve pathways.The presence of oral bacteria in systemic circulation is usuallyexpected when heavy bacterial plaques are present.

Alzheimer's disease is characterized by salient inflammatory features,microglial activation, and increased levels of proinflammatory cytokineswhich contribute to the inflammatory status of the central nervoussystem. The present inventors believe that, like other spirochetes baseddiseases, treatment decades in advance of the appearance of symptoms isat the heart of addressing Alzheimer's disease. For example, in variousways, Alzheimer's disease shares certain similarities with syphilis.Treponema pallidum (T. pallidum) persists in the syphilitic brain, whichsustains chronic infection and inflammation, forms amyloid plaques andcauses slowly progressive dementia. Dementia develops years or decadesfollowing the primary syphilitic infection. Historic observations andillustrations published in the first half of the 20th Century indeedconfirm that the pathological hallmarks, which define Alzheimer'sdisease are also present in syphilitic dementia. Cortical spirochetalcolonies are made up of innumerable tightly spiraled Treponema pallidumspirochetes, which are morphologically indistinguishable from senileplaques using conventional light microscopy. Local brain amyloidosisalso occurs in general paresis and, as in Alzheimer's disease,corresponds to amyloid beta. Thus, chronic spirochetal infections cancause dementia and support a causal relationship between variousspirochetal infections and Alzheimer's disease. They also indicate thatlocal invasion of the brain by these helically shaped bacteria reproducethe filamentous pathology characteristic of Alzheimer's disease. Chronicinfection by spirochetes, and co-infection with other bacteria, istherefore involved in the etiology of Alzheimer's disease. Addressingthis infection progression is one aspect of the present invention, andone focus is therefore on preventing spirochetes bacteria from growingin the oral cavity and in reducing the ability of such spirochetes totravel through the blood or via the nervous system to regions of thebrain.

The similarities of the clinical and pathological manifestations ofsyphilis and Lyme disease are well documented. Lyme disease, likeAlzheimer's disease, involves the creation of plaques in brain tissue.Thus, one aspect of the present invention is to adopt a similartreatment regimen with respect to Alzheimer's disease as has been foundto be effective to combat syphilis and Lyme disease. Syphilis is easy totreat with penicillin, one of the most widely used antibiotics. Peoplewho are allergic to penicillin may be treated with a different oralantibiotic, such as doxycycline, azithromycin, or ceftriaxone. Likesyphilis, however, the damage already done by Alzheimer's disease cannotbe reversed. It is thus imperative that early diagnosis, e.g. ofperiodontitis, be followed up with an appropriate treatment plan toavoid the dire consequences of Alzheimer's disease progression.

There is compelling evidence that treponemes, spiral-shaped bacteria,are involved in the etiology of several chronic diseases, includingchronic periodontitis. Treponemes are members of the normal oralmicrobiota of healthy individuals, albeit in very low numbers.Treponemes are members of the Spirochaetes phylum, a clade now believedto be distinct from both Gram-positive and Gram-negative bacteria, thatis believed to have undergone extensive horizontal gene transfer withArchae and possibly with eukaryotic organisms. Chronic periodontitis isa polymicrobial disease, and co-infection of Treponema denticola withother periodontal pathogens can enhance alveolar bone resorption. Thebacterium has a suite of molecular determinants that enable it to causetissue damage and subvert the host immune response. The human oralcavity harbors more than 60 different Treponema species, previouslyconsidered to be commensal spirochetes, but now revealed to bepredominant and invasive periodontal pathogens. Spirochetes frequentlyco-infect with other bacteria and viruses.

When considering the virulence characteristics of spirochetes, and inparticular, T. denticola, it is imperative to understand that it is partof a pathogenic bacterial consortium, and its interactions with otherbacterial species are important for disease pathology. The bacterialcomposition of subgingival plaque in individuals with chronicperiodontitis often find P. gingivalis and T. denticola and T. forsythiatogether.

Uncontrolled inflammation of the periodontal area may arise when complexmicrobial communities transition from a commensal to a pathogenicentity. Communication among constituent species leads to polymicrobialsynergy between metabolically compatible organisms that acquirefunctional specialization within the developing community. Keystonepathogens, even at low abundance, elevate community virulence, and theresulting dysbiotic community targets specific aspects of host immunityto further disable immune surveillance while promoting an overallinflammatory response. Inflammophilic organisms benefit fromproteinaceous substrates derived from inflammatory tissue breakdown.Inflammation and dysbiosis reinforce each other, and the escalatingenvironmental changes further select for a pathobiotic community.

There is a long felt but unsolved need for an answer to the Alzheimer'sdisease quandary. Without understanding the cause of the disease,however, attempts to simply combat symptoms are ill advised andpotentially dangerous. The treatment for AD may not be a blockbusterdrug that will entice large pharmaceutical companies. A silver bulletfor AD may therefore not be in the cards. Rather, as AD is a diseasethat is slow to progress due to its origins in an individual's oralmicrobiome and later progression to brain tissue, its treatment andprevention, as described herein, is achieved via methods and systemsthat are directed to improving the oral microbiome health, in theprevention of the occurrences that lead to the spread of oral bacteriato an individual's brain, and in the ongoing maintenance of oral healthvia use of novel oral strips that provide and/or nurture an environmentwhere beneficial bacteria can thrive and where spirochetes growth can becontrolled. Regular dental practices must be reviewed and adapted toprevent the occasions where harmful bacteria can reach a person'sbloodstream, and protective measures are warranted in suchcircumstances, followed by establishment of better oral microbiomeconditions to thwart the progression of AD. Lessons from the Amish canbe gleaned from their oral microbiome which appears to protect them frommany of the ravages of modern diseases.

In particular embodiments, addressing an individual's oral health in amanner directly related to brain health is achieved by various methods,including the purposeful exposure of an individual with bacteria of thegenus Prevotella to decrease the incidence of periodontitis, and theprovision of oral strips having antibacterial characteristics, etc. toreduce the likelihood of Alzheimer's Disease. Bacteria from the oralcavity can weaken the blood brain barrier, enter the brain, andaccelerate the events leading to cognitive decline and Alzheimer'sdisease. The present inventors submit that maintaining the health of theoral cavity offers an incalculable potential return on investment.

Various aspects of the present invention are directed to a method ofreducing the likelihood of an inflammatory disease, and in particularAD, developing in a subject, with such method including the provision ofa particularly designed bioadhesive strip to a subject that has beendiagnosed with periodontitis. Suitable strips are adapted for oralcavity application and have a first and second side, with the first sidehaving a bioadhesive that is adapted to bind to a portion of anindividual's oral cavity, and in particular either to dental surfaces ora mucosal membrane for an extended period of time, e.g. for at least 1hour, more preferably more than 3 hours, and in some scenarios, for atleast twelve hours or more, while inside a person's mouth. The secondside preferably has a specially textured surface that has ananti-microbial characteristic derived from its surface topography, suchtopography resisting bioadhesion of undesired bacteria that aretypically present in a human's mouth. Moreover, as spirochetes employcopper and manganese for growth, the strip is preferably devoid ofmanganese and copper. Conversely, because zinc is important to manybeneficial oral bacteria, such strips preferably include zinc componentstherein. As described herein the use of a strip that has on its surfacea pattern defined by a plurality of spaced apart features each having atleast one microscale dimension and having at least one neighboringfeature having a substantially different geometry, is useful in thatsuch a micro-surface design thwarts the growth of bacteria on itssurface. Use of such anti-bacterial textured surfaces is thereforeimportant in the maintenance of desired oral bacterial populations andin the ability, especially without the use of antibiotics, to reduce thegrowth of undesired bacteria, especially those associated withperiodontitis. Such strips can also or alternately include variousmineral (e.g. zinc) and anti-biotic components, such a variety ofantibiotics that preferably target undesired bacteria believed to beinvolved in the progression of periodontitis. As the formation ofbiofilms is believed instrumental in the ability of certain bacteria toavoid antibiotic contact, e.g. spirochetes, the use of agents thatassist in breaking down biofilms are important constituents of variousembodiments of the present invention. Thus, as one of skill in the artwill appreciate, a variety of anti-biofilm agents can be included in theoral strips as described herein, that may rely upon a variety ofanti-adhesion-mediated mechanisms. For example, curcumin, enzymes, likeDNase I, α-amylase and DspB, serrapeptase, etc can be employed.

Certain preferred embodiments include administering to the oral cavityof a subject an effective amount of Prevotella intermedia, even morepreferably Prevotella intermedia modified using CRISPR-Cas orCRISPR-Cpf1 to remove at least one virulence factor. It is believed thatmaintaining Prevotella bacteria in the oral microbiome, but having astrain that lacks particular virulence factors, such as those associatedwith biofilm formation, provides benefits in that the native ability ofsuch bacteria to produce NO is preserved, thus facilitating theretention of positive attributes while deleting undesiredcharacteristics of the bacteria. While Prevotella is one preferredspecies of bacteria that is believed useful in modifying in this regard,one of skill in the art will appreciate the various other bacteria thatreside in a person's oral microbiome that can also be modified togenerate and foster a microbiome that is more conducive to treatmentswith anti-biofilm formation, that permits the use of various antibioticsas described herein, and in establishing a better microbiome environmentsuch that spirochetes growth and infection can be addressed. The use ofthe oral strips as described herein as both a way to administeranti-bacterial substances and anti-biofilm agents, may also be employedto deliver desired amounts of desired bacteria, such as modifiedPrevotella, thus ensuring that such modified bacteria are placed in adesired portion of one's oral cavity to achieve maximum effect.

Methods of the present invention further include orally administering tothe subgingival tooth area of the subject an effective amount of anantibiotic effective to kill spirochetes bacteria residing on thesubgingival tooth area of the subject. Preferably the antibioticcomprises one of doxycycline and methotrexate. Moreover, such stripspreferably include between 0.2 and 0.9% xylitol by weight. Also, suchstrips may further include an effective amount of paquinimod such thatwhen applied to a tooth area of the subject, is effective to inhibitcollagenase activity. In still other embodiments the strip comprises oneof bioluminescent material, compounds that facilitate the growth ofdesired bacteria beneficial to a person's health, and/or at least about200 mg. xylitol. One will appreciate that the strips can have variousconstructions and structures to facilitate functional aspects sought tobe achieved. For example, certain embodiments employ strips havingencapsulated materials that can elute from the strip at particular timesor via the purposeful rupture of an encapsulated (e.g. frangible bypressure) structure so as to cause the release of an encapsulatedmaterial or composition. In some embodiments, a strip havingencapsulated pockets may include one of Prevotella, iron, NO,antibiotics, anti-biofilm agents and/or anti-inflammatory agents, etc.thus rendering it possible to apply a customized strip to a person'soral cavity to effectively address the particular situation.

Porphyromonas gingivalis is one of the major etiological agents involvedin advanced adult periodontitis and its virulence factors, such aslipopolysaccharide, fimbriae, haemagglutinins, vesicles and proteases,have been characterized. Among these virulence factors,arginine-specific cysteine proteinase (Arg-gingipain, Rgp) andlysine-specific cysteine proteinase (Lys-gingipain, Kgp), whichspecifically cleave synthetic and natural substrates at the carboxylsides of arginine and lysine residues, respectively, have receivedconsiderable attention due to their strong ability to degrade a broadrange of host proteins. Rgp and Kgp activities cause not onlydestruction of periodontal tissue but also disruption of host-defencemechanisms. Rgp is involved in fimbriation.

The initial step of Por. gingivalis attachment to the oral tissue hasbeen shown to be fimbriae-mediated and the attachment can be achieved byPor. gingivalis adherence to micro-organisms that have already colonizedthe periodontal regions. Por. gingivalis can co-aggregate withPrevotella intermedia, which is one of the early colonizers inperiodontal niches. Co-aggregation among bacterial cells caused by theadherence of one bacterial species to another is a colonizationmechanism. There are several aggregation factors for co-aggregationbetween Porphyromonas (Por.) gingivalis and Prevotella (Pre.)intermedia. Moreover, as part of the infective process, Porphyromonasgingivalis must acquire heme which is indispensable for life and enablesthe microorganism to survive and multiply at the infection site. A hmuheme uptake system with a leading role played by the HmuY hemophore-likeprotein, is responsible for acquiring heme and increasing virulence ofthis periodontopathogen. Prevotella intermedia produces two HmuYhomologs, termed PinO and PinA.

Several virulence factors contribute to differences in the bacterialvirulence of P. gingivalis, in particular the Fimbriae andhemagglutinins. Based on the diversity of the fimA gene, fimbriae areclassified into six genotypes (I-V and Ib) (Hamada et al., 1994;Nakagawa et al., 2000), being type II fimbriae, followed by type IV, themost frequently found in P. gingivalis strains isolated fromperiodontitis patients (Amano et al., 2000, 2004; Missailidis et al.,2004). In contrast, healthy adults carry strains coding type I fimA(Amano et al., 2000). P. gingivalis possesses at least eighthemagglutinins encoded by the hag genes (hagA to hagE) (Progulske-Fox etal., 1989, 1995; Nelson et al., 2003). Among them, the hemagglutininsHagA, HagB, and HagC have been described as important virulence factors(Bélanger et al., 2012; Connolly et al., 2017). A number of virulencefactors have been reported to contribute to the pathogenicity of P.gingivalis including; LPS, fimbriae, hemagglutinin, hemolysin andextracellular hydrolytic enzymes (especially the Arg-X and Lys-Xspecific proteinases), otherwise known as “P. gingivalis trypsin-likeenzymes”.

Porphyromonas gingivalis, a keystone pathogen of periodontal disease,has been reported to be associated with human Alzheimer's Disease (AD).The inflammatory burden following oral infection and entry of thisbacterium into the brain appears to drive the formation of amyloid-betaplaques and neurofibrillary tangles with loss of cognition. P.gingivalis is a master of immune subversion in this inflammatory cascadeand may establish microbial dysbiosis where it is located.

Gingipains are involved in AD in that they are believed to enhance theeffect of polymorphic complement gene defects, allowing for a localinfection and the recruitment of additional bacteria that are resistantto the bactericidal activity of complement.

An ideal window to control the impact of peripheral inflammation fromperiodontitis on AD is at the time of diagnosis of the oral disease. Theclinical value of inhibiting all the three main pathways of complementactivation was recently suggested in periodontitis and can be achievedby targeting the central component C3, which directly inhibitsinflammation and indirectly counteracts dysbiosis. Gingipain inhibitors(Kgp) are valuable for treating P. gingivalis colonization of the ADbrain.

To further comply with written description and enablement requirements,the following are incorporated herein by reference: 20190315642 toParsley; 20170246269 to Hajishengallis; 20130236488 to Dashper; and20030083287 to Burgess.

Orally administering to the subject after at least 24 hours afterremoval of said strip from the subject a pharmaceutically effectiveamount of a preparation comprising Prevotella intermedia bacteria thatproduces nitric oxide is believed to be effective in that such an oralbacterial population mimics that observed in the Amish populations wherethe incidence of AD is rare. Yet further methods involve the provisionof a strip to a person who has been diagnosed with periodontitis andincludes use of a strip that includes an effective amount of Prevotellaintermedia that has been modified to remove at least one virulencefactor, as well as at least about 200 mg. xylitol and an effectiveamount of paquinimod to inhibit collagenase activity.

These and other aspects of the present invention are described herein,offering for the first time, both a causative explanation for AD, aswell as practicable and beneficial ways to reduce its progression.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a photograph of Dr. Alios Alzheimer for which the diseasesought to be treated via the present invention is named after.

FIG. 2A-2D illustrate various illustrations of oral strips that havemicro surfaces that deter bacteria from residing on such surfaces.

FIG. 3 illustrates a CAT scan section of a human brain showing evidenceof Alzheimer's disease.

FIG. 4 shows close up views of spirochetes, illustrating their spiralnature and conformation.

FIG. 5 illustrates oral biofilm infections of the human body, showing ahuman tooth, gum and the sites of periodontal disease.

FIG. 6 illustrates a site of periodontal disease and to areas of thehuman body believed to be causally affected by inflammation andbacterial infections stemming therefrom.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE PRESENT INVENTION

FIG. 1 shows a photograph of Dr. Alios Alzheimer for which the diseasesought to be treated via the present invention is named after. It isinteresting that the prevalence of AD is so recent. Discovered in 1906as a rarity, AD has now become one of the top health concerns in theworld. The rise in AD raises questions as to what present practices arecausing its progression. As described herein, while preventative dentalhealth is one way in which to combat AD, the present inventors alsobelieve that the rise of dentistry during the last 100 years has alsoplayed a causative role in AD in that it is much more common today for aperson to engage in activities where their gums bleed, thus providing aroute for various oral microbes to enter the blood stream and causevarious diseases, including AD, arteriosclerosis, some types ofinflammatory arthritis, etc.

According to various aspects of the present invention, one criticalappreciation relates to the inventors' contention that bacterialinfections are at the heart of many of today's chronic diseases. Forexample, it is believed that periodontal disease is a risk factor forcardiovascular disease and stroke, as well as a host of other diseasesmentioned herein, including Alzheimer's disease, IBD, Crohn's Disease,etc. Because periodontal disease is a modifiable risk factor that can beprevented and treated—the long felt but unsolved treatments for variouschronic diseases can also be prevented and treated. Thus, employing acombination of a restorative microbiome approach and an antimicrobialapproach, provides for the implementation of treatment that isspecifically designed to improve a person's periodontal condition so asto reduce and/or delay future chronic disease, with Alzheimer's diseasebeing one of many. It is believed that one of the reasons such anapproach has not earlier been adopted is that when it comes to dentalhealth, there exists several centuries of dental teaching that statesthat periodontal disease results from a “dirty mouth.” Thus, suggestingthe use of microbes in the oral cavity to alleviate disease may appearto many as being counterintuitive and as a “teaching away” from longheld beliefs and practices.

Periodontal diseases are very common, affecting up to 90% of the globalpopulation. The common inflammatory forms of the diseases are caused bypathogenic microflora in the biofilm or dental plaque that formsadjacent to the teeth on a daily basis and can result in loosening ofteeth, occasional pain, and eventual tooth loss. Several Gram-negativebacteria, including Porphyromonas gingivalis, Treponema denticola, andTannerella forsythia, are frequently isolated from dental plaques inperiodontal patients and were initially considered periodontalpathogens. A strong correlation between several cultivable bacteria suchas Prevotella intermedia, Fusobacterium nucleatum, Aggregatibacteractinomycetemcomitans, and Eubacterium nodatum and periodontal diseasehas also been reported. Although subgingival bacteria are the majorcause of periodontal diseases, more than one-half of subgingivalbacterial species or phylotypes are not readily cultivable, whichpresents an obstacle to fully understand the causal relationshipsbetween subgingival bacteria and periodontitis. To overcome thedifficulties and limitations associated with cultivation,culture-independent methods based on amplification and sequencing ofbacterial metagenomes have been developed to identify thousands ofdifferent bacteria in a single sample.

There are two main forms of periodontal disease: gingivitis andperiodontitis. Both are pathologic periodontal inflammatory processesthat are the result of an accumulation of dental plaque. The most commonis the plaque-induced gingival disease called gingivitis, which is areversible form of periodontal disease. The other form of periodontaldisease is periodontitis, a destructive, irreversible, chronicinflammatory process due to dental plaque, which results in atrophy orloss of the underlying bone and connective tissue support around theteeth. Periodontitis is the most common chronic inflammatory diseaseknown to mankind.

The present inventors believe that effective management of periodontitisis a key to the prevention of Alzheimer's disease, as periodontitis isboth treatable and preventable. Thus, oral health is directly related tobrain health and with purposeful reduction in the numbers of certainbacteria and exposure to certain bacteria species, such as in certainembodiments, Prevotella, the benefits of decreased periodontitis, andthus, the reduction in the emergence of Alzheimer's disease, can beachieved. Prevention of Alzheimer's disease through the use ofmicrobiome products is believed to be one of the best ways to avoid theprevalence of Alzheimer's disease and the public expenses related to itsmanagement.

Periodontitis is a common oral infection associated with primarily gramnegative anaerobic bacteria. Periodontitis is an inflammatory diseasecaused by a microbial biofilm, characterized by periodontal pocketformation, attachment loss and loss of supporting alveolar bone.Periodontally compromised teeth lose function and may have to beextracted, which often requires costly prosthetic rehabilitations. Inindustrialized countries, approximately 50% of the adult populationsuffers from moderate or severe periodontitis.

Dental plaque is unlike any other bacterial ecosystem. Plaque is dividedinto two distinct types based on the relationship of the plaque to thegingival margin, i.e., supragingival plaque and subgingival plaque. Thesubgingival plaque harbors an anaerobic gram-negative flora has beenassociated with Periodontitis. Periodontitis can be marked as a“low-grade systemic disease” by release of proinflammatory cytokinesinto systemic circulation and elevation of C-reactive protein (CRP).

Although bacteria are involved in periodontal infections, it is not thescenario of a typical infection, as the offending bacteria generallyremain outside the body, attached to the tooth. When gum disease reachesa bleeding stage, the bacterial components of the plaque changes andthere is an increase in various species, including Prevotella species,which have nutritional requirements derived from the host, suchnutrients becoming available as a result of the tissue inflammation andbleeding.

While not bound by theory, Prevotella in an individual's oral cavity,due to its production of nitric oxide, plays a beneficial role inmaintaining oral health in a fashion that prevents AD progression. WhilePrevotella is also seen in biofilms that may ultimately also be involvedin providing conditions for spirochetes to be protected and grow, andultimately to be released into the blood or along nerves to reach thebrain, the presence of Prevotella should not automatically be consideredas a negative. The conventional thinking that various of the bacteriafound in one's oral microbiome are clearly categorized as either “good”or “bad” is outdated and largely inaccurate. Depending upon otherconditions, the presence of particular bacteria previously deemed to bepathogenic, may indeed serve a beneficial role in maintaining a person'soral microbiome health, including in establishing a better oralmicrobiome to prevent the progression of AD.

While there are over 500 species of microbial species that are believedto be primary etiologic agents for periodontal disease, only arelatively small number most frequently associated with activeperiodontal disease, including gram negative, anaerobic pathogens:Porphyromonas gingivalis, Tanneralla denticola, Tanneralla forsythia;Fusobacterium nucleatum, Prevotella intermedia, Prevotella nigrescens,Peptostreptococcus micros, Campylobacter rectus, Centruroides gracilis,Campylobacter showae, Eubacterium nodatum, and Streptococcusconstellatus. Thus, only a limited number of gram-negative anaerobes aresignificantly associated with periodontal disease. Various embodimentsof the present invention are focused on the more refined selection of anappropriate drug to employ to combat particular gram negative, anaerobicpathogens.

Current treatment for periodontitis involves removal of all bacteriafrom the subgingival pockets. Removal of subgingival plaque by currenttreatment methods is temporary, since the subgingival packet may bere-colonized after cleaning by organisms from the supragingivalreservoir. For individuals suffering periodontal disease, currentpractice also involves the use of antimicrobial agents that aretypically chosen to kill as many bacterial types as possible, oftenemploying broad-spectrum agents such as tetracycline, amoxicillin andmetronidazole, and often leading to the overuse of these agents. Whileuse of antimicrobial agents plays a part in various embodiments of thepresent invention, such use is more nuanced and the destruction ofbeneficial bacteria in the oral cavity is either prevented orreestablishment of such bacteria is fostered so as to maintain a healthyoral microbiome that can prevent periodontal disease, and thus alsoprevent the onset of Alzheimer's disease. The uncritical use ofantibiotics could increase bacterial resistances. One goal of thepresent invention is to preserve the maintenance of an ecologicallybalanced biodiversity of the microflora within the oral cavity as it iscrucial not only to the oral health but also to the general health ofthe individual, especially in avoiding Alzheimer's disease.

Surprisingly, clinicians have been advised that they do not really haveto test for what microbe might be involved in periodontitis, and insteadmay simply use antibiotics on an empirical basis to see how they mightwork. Thus, one aspect of the present invention is to halt suchindiscriminate use of antibiotics—and to have the clinician carefullyevaluate the presence of specific gram positive anaerobic bacteria thatcause periodontitis, and more importantly, to re-administer beneficialbacteria to the oral cavity after antibiotic treatments. Indeed, currentpractice for the treatment of chronic periodontitis includes arecommendation to use more than one antibiotic with differentantibacterial spectra under the misguided belief that a broad diversityof periopathogens must be killed, including anaerobic, microaerophilic,and aerobic bacteria, both Gram negative and Gram positive. One aspectof the present invention is to target particular gram negativeorganisms, specifically not including other organisms, such as A.Actinomycetemcomitans, as the use of antibiotics against this organismis believed to hinder, rather than promote, the objective ofestablishing a healthy microbiome that prevents Alzheimer's disease.

The progress in finding treatments for periodontal disease has beenhindered in that there has been no consensus as to whether an anaerobicor microaerophilic infection is involved. Only recently have studiesshown that A. actinomycetemcomitans is not, as was previously assumed,an important periodontopathogen in major periodontal diseases. Itsreintroduction after antibiotic treatment is therefore one aspect ofseveral embodiments of the present invention.

A broad based antibiotic therapy as part of an early treatment regimenin patients diagnosed with Alzheimer's disease, without an appreciationof the wider beneficial aspects of the oral microbiome—could lead to aworsening of Alzheimer's disease, rather than a treatment for it. Asdescribed herein, a similar concern relates to destruction of biofilmswithout an appreciation of how spirochetes may thus be freed to inflictfurther damage. Indeed, in various embodiments of the present invention,there is a fostering of the establishment of certain oral bacteriapopulations, e.g. Prevotella bacteria, etc. which would be killed byindiscriminate use of antibiotics.

Treatment for patients in the early stages of dementia may require morethat the administration of antibiotics, such as penicillin, aspenetration through oral biofilms necessitates the use of an effectiveagent to disperse the biofilm. Such anti-biofilm agents include, forexample, furans (citalopram), thiophenes (olanzapine), piperidines(donepezil), pyrroles (azoles), [and rifampin. Donepazil (ananticholinesterase inhibitor and also a biofilm disperser) is apreferred anti-biofilm agent for incorporation into the oral strips ofthe present invention such that it can elute directly onto gum antissue, thus avoiding the need for systemic administration of drugs thatwould have deleterious effects on other parts of the body not beingtreated for biofilm formation.

Importantly, while biofilm formation in the oral cavity is sought to bedisrupted so as to enable the killing of undesired bacteria, thedispersal of biofilm formation in a person's brain must be consideredwith special caution. This is so because dispersal of a biofilm and therelease of spirochetes bacteria form such biofilm, may result in theunintended spread of the spirochetes to other regions where similarbiofilm and plaque buildup may occur. The dispersal effect in such caseswould therefore potentially create many more plaques, e.g. the use ofhaloperidol in treating AD has been shunned. Thus, in certainembodiments, and especially for early dementia, penicillin is preferablyadministered, in some situations both systemically and in the moreconcentrated strip contacting form, In treatment of the “latent” stageof AD, the use of penicillin or other suitable antibiotics is advisable,but with the caveat that disruption of the biofilm and plaque structuresshould be carefully considered so as not to spread spirochetesthroughout the body and brain tissue. Dosages, etc. for antibioticswould be similar to those employed in the treatment of latent syphilis.Moreover, treatment of a person with appropriate antibiotics prior toany dental surgery is recommended to avoid unintended release ofspirochetes into a person's bloodstream. This entails treating thespirochetes before they reach the brain in the case of dental surgeryand before they do damage or get caught in biofilms or plaques in latentdisease.

Combinations of antibiotics may be employed to enhance the effect ofindividual antimicrobials through synergic interactions and for thetreatment of biofilm-associated infections. For example, rifampin,vancomycin and fusidic acid may be used in combination with other listedantibiotics to achieve the control of spirochetes infections, especiallyvia the incorporation of these agents in the strips as described hereinas an effective way to disperse biofilms and render antibiotics moreeffective against spirochetes and associated bacteria in combatingvarious biofilm-associated diseases. Various antibiotics can be employedwith the strip embodiments of the present invention, including but notlimited to: daptomycin, daunomycin, doxorubicin, and mitomycin C thathad good activity against B. burgdorferi; and plant-made antimicrobialpeptides such as retrocyclin and protegrin.

In various aspects of the present invention, the disruption of biofilmsis achieved via one or more agents that are preferably incorporated intoa strip for use in the oral cavity of a person. By even partiallydisrupting the biofilm structure, the remaining in the oral cavity andespecially those bacteria contributing to a biofilm, can become morevulnerable to antimicrobial agents. Therefore, substances that affectbiofilm biomass are believed to be of considerable utility for thetreatment of biofilms in that disruption thereof can assist in treatingspirochetes infection in the oral cavity before spirochetes are able totravel to a person's brain tissue. In certain preferred embodiments, oneof xylitol and famesol are employed to facilitate degradation of abiofilm in the oral cavity. In still other embodiments, sodiumhypochlorite is used as an anti-biofilm agent, preferably in at leastabout a 6% concentration. In addition, there are many commonlyprescribed medications that have additional abilities to act as systemicbiofilm dispersers. These include piperidines: (donepezil, haloperidol,risperidone), thiophenes (olanzapine), furans (citalopram), and pyrroles(leflunomide, itraconazole, celecoxib, and atorvastatin).

In various embodiments, one aspect of the present invention is directedto therapies to treat or prevent the onset of periodontal disease, whichin some embodiments that target agents that inhibit the adherence of P.gingivalis to supragingival plaque, such as can be included in mouthrinses and toothpaste formulations, so that they may be easily andnon-invasively administered. P. gingivalis gains systemic exposurethrough damage to gingival tissues. Therefore, while not bound bytheory, one aspect of the present invention relates to limiting P.gingivalis adherence to supragingival plaque in the oral cavity has adramatic effect on systemic diseases, including atherosclerosis andAlzheimer's disease.

In preferred embodiments, the prevention of the growth of particularpathogenic organisms is achieved without destruction of other helpfulorganisms that inhabit the oral cavity. In certain embodiments, agentsare employed to prevent particular biofilms, such as P. gingivalisbiofilms, to establish an oral microbiome environment that will halt theprogression of Alzheimer's disease. Incorporated herein by thisreference in their entireties are the following US patents and patentpublication nos.: 20120142548 to Corsi et al.; U.S. Pat. Nos. 6,287,610,6,569,474, US20020009520, US20030206995, US20070054008; and U.S. Pat.No. 8,349,313 to Smith; and U.S. Pat. No. 9,011,834 to McKenzie.

FIG. 2A-2D illustrate various illustrations of oral strips that havemicro surfaces that deter bacteria from residing on such surfaces. Inparticular embodiments of the present invention, an oral strip,preferably mucoadhesive in nature and preferably with xylitol therein,is employed to apply a locally acting collagenase inhibitor agentXylitol, for example, included with such strips, reduces bacteriagrowth. Other oral strip embodiments include the provision of activeagents in addition to (or not) the antibacterial surface structures asdescribed herein as illustrated in FIGS. 2A-2D. After applying suchstrip to the oral cavity, it is able to release a preferred agent ordrug slowly as it is dissolved, so that the drug concentration in salivaexists for an extended time and maintains the desired inhibitoryconcentration. Blood and saliva are similar in many respects, but due toblood being conveyed outside the oral cavity, it poses a significantroute for bacteria to infect various other portions of one's body.Treating oral bacteria while in the oral cavity is therefore a preferredway to address bacterial populations so as to control their detrimentaleffects if and when they escape the oral cavity. Altering the conditionsin a person's oral cavity so that a preferred population of oralbacteria are fostered is one aspect of the present invention. The oralstrips as described herein may include various agents, such as xylitol,antibiotics, beneficial bacteria, etc. that provides for the ongoing andlow dose concentrations required for certain bacteria to thrive andothers to not thrive. Moreover, inclusion of encapsulated pockets insuch oral strips provides a way to introduce preferred bacteria into theoral cavity. Thus, providing modified Prevotella bacteria (e.g. modifiedso as to reduce or hinder various virulence facts of a nativePrevotella) is one way in which to encourage the growth of bacteria in aperson's oral cavity in a manner that is believed to be beneficial indeterring the progression of AD.

Such antibacterial structures as illustrated in FIG. 2A_D interfere withthe formation of biofilms that would otherwise form in a person's oralcavity. By disrupting biofilm formation, one is able to hinder theprogress of biofilms that are associated with the progression ofperiodontitis and in such a way, interfere with the progression of AD.The coating of dental surfaces—as well as the mucous membranes insidethe oral cavity—using the anti-bacterial surfaces as described herein,and in particular those as described in one of the parent applicationsto the present case, may be employed to further reduce the number andtype of undesired bacteria in the oral cavity.

Use of the strips of the present invention also overcomes certainproblems with conventional formulations of doxycycline hydrochloride,including undesired gastrointestinal reactions that produce systemictoxicity, and further eliminates complications arising from the use ofdrug infused chips, which are inserted into periodontal pockets as wellas other resorbable gel compositions, which provide forcontrolled-release of doxycycline for approximately one week. AsPeriostat® requires twice daily dosing and raises concerns about patientcompliance, the strips of the present invention are believed to besuperior and highly beneficial in terms of compliance and effectiveness,without overdosing of drugs in a manner that may cause problems stemmingfrom the long-term administration of antibiotics and consequentialreduction or elimination of healthy biotic flora, such as intestinalflora, which can lead to the production of antibiotic resistanceorganisms or the overgrowth of yeast and fungi. Thus, the strips of thepresent invention provide a unique way to achieve the benefit ofanti-collagen destructive enzymes while avoiding other undesiredantibacterial effects.

Alzheimer's disease was thought to be a disorder related to synthesisand decline in the degradation of AβP. FIG. 3 illustrates a CAT scansection of a human brain showing evidence of Alzheimer's disease. Thereis recent and compelling evidence that Aβ, however, is not simply amisfolded protein that accumulates in the brain, but is instead aprotein with physiological roles that responds to several pathologicalcontexts. The pathological process of Alzheimer's disease is thought tobegin long before the diagnosis of dementia is made and thus, anappropriate targeted treatment should start early in order to preventdementia.

Periodontal disease is an inflammatory disease in which the inflammatoryresponse, followed by the acquired immune response, drives thepathogenesis of periodontal tissue destruction. Periodontal pathogens,which are more or less universally present in low numbers, useinflammation to provide an environment to foster their growth. While fora long time it was thought that bacteria was the factor that linkedperiodontal disease to other diseases in the body, more recent researchdemonstrates that inflammation may be responsible for the association.One aspect of the present invention is directed to the appreciation thatpreventing and treating inflammation involved in periodontitis (and theresultant increase in host-derived, tissue-destructive enzymes, e.g.,collagenase plus other matrix metalloproteinases (MMPs), will help withthe management of other chronic inflammatory conditions, includingAlzheimer's disease.

The foundational characteristics of all inflammatory diseases is theup-regulation of cytokines, prostaglandins, MMPs (i.e. host derived,tissue-destructive matrix metalloproteinases), reactive oxygen species,etc. A major event in the link between local periodontitis and relevantsystemic/medical conditions is the release, from the inflamedperiodontal tissues of inflammatory mediators into the bloodstream,which subsequently travel to the liver. Once inflammatory mediators arepresent in the blood (i.e. derived from the inflamed gingiva), the liveris stimulated to produce acute phase proteins, which are diagnosticmarkers and mediators of inflammatory disease; one being C-reactiveprotein (CRP). To add insult to injury, LDL (low density lipoprotein)cholesterol, when oxidized by the inflammatory response, then forms achemical reaction with CRP. The end result is a complex of oxidized LDLcombined with CRP, which is taken up by macrophages in the atheroma andthese macrophages differentiate into foam cells, found in lipid-ladenplaques in the arteries and that are associated with increased risk ofheart attack and stroke. The foam cells, in turn, release MMP's, such asMMP-8, also known as collagenase. Collagenase's primary function is tobreak-down collagen. A collagen rich protective cap that encapsulatesatherosclerotic plaque is thus destroyed by collagenase, often leadingto a thrombosis, followed by stroke or a heart attack. Thus, while aprincipal focus of the present invention is on the prevention ofAlzheimer's disease, one of skill in the art will appreciate the variousother important diseases may also be addressed via the guidance providedin the present specification, and such other aspects should beunderstood as also being a part of the present invention.

While the human body is continually destroying old collagen, followed bya renewal process of normal turnover, in chronic inflammatory disease,collagenases, particularly MMP-8, become excessive and the normal repairprocess is halted. The inflammatory mediators (cytokines, prostaglandin,MMPs) present with oral inflammation, flow into the bloodbi-directionally from the gingiva into the circulation, resulting insystemic inflammation. Thus, unless there is a cessation of theinflammatory response, treatment is rendered difficult, if notimpossible.

Slowing down the breakdown of collagen and/or inhibiting the productionof collagenase is thus one aspect of the present invention and is oneway in which to prevent the progression of Alzheimer's disease. Collagendegradation can be inhibited, for example by using TIMP proteins (tissueinhibitors of metalloproteinases). To reduce potential degradation ofpolypeptides, certain tripeptides may be employed as collagenaseinhibitors, as well as other collagenase inhibitors, e.g. such aspaquinimod, and those one of skill in the art will appreciate can beemployed for the present purposes, see e.g., U.S. Pat. Nos. 4,687,841and 4,720,486 to Spilburg, et al.; US patent publication no. 20070207955to Tanihara; all incorporated herein by this references).

It is known that tetracyclines, a class of drugs that had previouslybeen recognized only as antibiotics, were unexpectedly found to blockcollagenase in mammals. This lead to the development of a formulation ofdoxycycline that inhibits collagenase (MMP-8) and other MMPs at a bloodlevel so low that it would NOT perform as an antibiotic known under thetrade name, of Periostat®. Low dose or subantimicrobial dose doxycyclineis the first systemically administered collagenase inhibitor drugapproved by the U.S. FDA. In particular embodiments of the presentinvention, a topical and locally acting collagenase inhibitor agent,similar to doxycycline, is employed to treat periodontitis, which willact to prevent the progression of Alzheimer's disease. In oneembodiment, an individual at risk of further developing periodontitis isadministered doxycycline plus methotrexate (MTX), preferably a low dose(about 20 mg) of doxycycline twice daily with MTX.

One aspect of the present invention is to provide antimicrobial agentsformulated into oral care products to augment mechanical plaque control.A delicate balance is needed, however, to control the oral microbiota atlevels compatible with health, without killing beneficial bacteria andlosing the key benefits delivered by these resident microbes. In certainembodiments, the strips as described herein are employed or thispurpose. Importantly, in various embodiments of the invention, insteadof focusing on the destruction of one particular species of bacteria,the native bacterial populations of an individual are adapted to removeparticular virulence factors from such a species. The destruction of thenative oral bacteria and the replacement of such bacteria with the abovedescribed modified bacteria, e.g. having impaired virulence facts, isone way in which to reduce the formation of undesired biofilms that canharbor spirochetes that are the root cause of AD. One of skill in theart, especially with the employment of CRISPR-Cas systems, is thereforeable to adapt native bacteria form a person's oral microbiome andtransform such a population to enhance the overall health of the oralcavity and in a manner that reduces the chances that AD will progress.

To comply with written description and enablement requirements,incorporated herein by the following references are the following patentpublications: U.S. Patent publication Nos. 2014/0349405 to Sontheimer;2014/0377278 to Elinav; 2014/0045744 to Gordon; 2013/0259834 toKlaenhammer; 2013/0157876 to Lynch; 2012/0276143 to O'Mahony;2015/0064138 to Lu; 2009/0205083 to Gupta et al.; 2015/0132263 to Liu;and 2014/0068797 to Doudna; U.S. Pat. No. 8,945,839 to Zhang;2014/0255351 to Berstad et al.; 2015/0086581 to Li; PCT/US2014/036849and WO 2013026000 to Bryan; 2016/0199424 to Berry et al.; 2013/0326645to Cost et al.; 2016/0206666 to Falb; 2017/0042860 to Kashyap and2015/0045546 to Siksnys.

One aspect of the present invention is directed to the maintenance of apopulation of Prevotella bacteria in the oral cavity that is similar topopulations found in healthy Amish individuals where Alzheimer's diseaseis a rarity. Amish individuals were found to have elevated levels ofPrevotella in their oral cavity as compared with other individuals.Prevotella is known to produce nitric oxide. Nitric oxide (NO) is knownto have potent antimicrobial properties and is an important cellularsignaling molecule. NO is a free radical with an unpaired electron.Although the earliest studies in the field suggested that NO is astrictly pro-inflammatory macrophage product, it is clear from thecurrent literature that, in fact, NO is made by numerous cell types andis often anti-inflammatory. Much of this dichotomy can be explained bythe particular responses of given cells involved in the inflammatoryresponse, but another variable involves the complex chemistry in whichNO can participate.

Nitric oxide is a ubiquitous intercellular messenger molecule withimportant cardiovascular, neurological, and immune functions. Nitricoxide is a short-lived, reactive free radical that participates in avariety of reactions and in small controlled concentrations in the body,it acts as a physiological and pathophysiological mediator and it playsan important role in biological systems. The assessment of the stableend products of NO, nitrite and nitrate (NOx), is commonly used as ameasure of the NO production in biological fluids. The production ofnitric oxide represents a mechanism of pathogen destruction in activatedneutrophils. Production of NO or expression of inducible NO synthase(iNOS) by peripheral neutrophils or in gingival tissues is associatedwith periodontal disease. The massive presence of neutrophils and theirenhanced activity at sites of periodontal disease have sparked debate asto whether neutrophils are responsible for the destruction ofperiodontal tissues or whether they play protective roles in controllingpathogenic bacteria involved in periodontal disease. Neutrophils fromperiodontitis patients produced significantly lower levels of NO levelswhen compared to neutrophils from healthy subjects. Low NO levels wereproduced by neutrophils from chronic periodontitis patients. So thepresence of NO seems to be desired in avoiding periodontitis. Prevotellais associated with increased NO production and thus, Prevotella—whilealso associated with periodontitis, is oddly believed to be beneficialto individuals. The recruitment of neutrophils and other leukocytes inthe periodontal pocket is an important feature of the inflammatoryprocess in periodontal disease. Neutrophils play an important role inperiodontitis by producing nitric oxide (NO) and antimicrobial peptides,molecules with microbicidal activity via oxygen-dependent and-independent mechanisms, respectively. The use of nitric oxide todisperse biofilms may be employed to improve infectious diseasetreatments. The use of low levels of NO to exploit its signalingproperties to induce dispersal represents an unprecedented and promisingstrategy for the control of biofilms in clinical contexts.

Prevotella, previously classified in the genus Bacteroides, is a genusof an obligate anaerobic gram-negative rod-shape bacterium. Althoughthey generally have a limited ability to ferment amino acid and requirehemin and menadione to grow, Prevotella is a versatile genus which hasbeen observed in various niches, such as oral cavity, upper respiratorytract, urogenital tract, rumen and human feces. Prevotella is alsowell-known as a preventative agent for the bovine disease of rumenacidosis. Rumen acidosis greatly affects milk production of cattle bydisrupting the typical digestive processes of the stomach. As anotherindication that Prevotella may be beneficial, this leads to an increasedsusceptibility to other pathogenic forces which also affect the healthof food provided from the cattle.

Periodontal diseases are chronic inflammatory infections associated withgram-negative bacteria which stimulate macrophages to generate NO.Prevotella intermedia has conventionally been considered to be one ofthe causative pathogens of periodontal disease. The increased populationof such bacteria in healthy Amish individuals, however, coupled with thescarcity of Alzheimer's disease in the Amish population, presents aclassic case where one of ordinary skill in the art of periodontitistreatment, would not promote the use of Prevotella.

Chronic inflammation is characterized by a proliferation of fibroblastsand formation of blood vessels (angiogenesis), as well as an influx ofchronic inflammatory cells, namely granulocytes (neutrophils,eosinophils, and basophils), lymphocytes, plasma cells and macrophages.Nearly two decades ago, the production of nitrogen oxides was associatedwith inflammation. The metabolic pathway known as the Larginine: NOpathway is the main source for the production of NO in mammalian cellsby a group of enzymes known as the nitric oxide synthases (NOS). Theenzyme primarily responsible for the roles of NO in inflammatoryprocesses is the inducible NOS (iNOS; NOS2; or type II NOS), which isnot typically expressed in resting cells and must first be induced bycertain cytokines or microbial products.

In recent years, NO has emerged as a major mediator of inflammation. Asmight be expected from such a pleiotropic molecule, there arecontradictory reports in the literature concerning its role as ananti-inflammatory or proinflammatory agent. The inconsistencies reportedprobably are due to the multiple cellular actions of this molecule, thelevel and site of NO production, and the redox milieu into which it isreleased.

The dichotomous role of NO in inflammation, often referred to as the NOparadox, is based mainly on the conflicting data showing the effects ofNOS inhibitors of varying selectivity in different animal models. Thephysiological and pathological functions of NO are diverse and oftencontradictory. NO acts as a useful endogenous free-radical scavenger. NOmay provide a chemical barrier to cytotoxic free radicals. NO may have aconsiderable protective effect on cellular viability and can act as anantioxidant protecting cells from oxidant-induced damage and preventingendothelial apoptosis. Low NO concentrations contribute to endothelialcell survival and high NO levels induce the apoptosis of endothelialcell. Any assessment of the role of NO in human disease must take intoaccount the dual role of NO.

Many of the regulatory and physiological functions of NO can beconsidered as protective or “anti-inflammatory,” and are mainly relatedto NO produced by the other isoforms of NOS. Other data exists, however,that iNOS expression is found in an increasing number of humandisorders, for example, nitric oxide is crucial in the pathogenesis ofseptic shock. It is believed that certain microorganisms have developedmeans for suppressing the expression and/or activity of iNOS, perhaps byco-opting the host's own regulatory machinery. Viewed from thisperspective, the balance between induction and suppression of iNOS mayunderlie much of the physiology and pathology of inflammation.Nonspecific inhibition of iNOS also has been reported to be detrimental,rather than beneficial. Release of NO has been reported in inflammatoryresponses initiated by microbial products or autoimmune reactions. Theeffects of NO on specific immunity is under investigation. Most of theexisting data suggest that NO suppresses, rather than enhances,lymphocyte activation and proliferation. One possible explanation forthese often contradictory results is that iNOS inhibition is detrimentalto the host during priming of pathogenic T-cell responses in theperiphery, but largely protective at the site of disease. It appearsthat NO plays an important role in the pathogenesis of chronicinflammation. Nitric oxide stimulates TNF-_ production bysynoviocytesand its catabolic effects on chondrocyte function promote thedegradation of articular cartilage implicated in certain rheumaticdiseases. Studies indicate that NO is at least partly responsible forIL-1-induced suppression of glycosaminoglycan and collagen synthesis. Inhuman chondrocytes, IL-18 has been identified as a cytokine thatregulates chondrocyte responses and contributes to cartilage destructionthrough stimulation of the expression of several genes, including iNOS,inducible COX, IL-6, and stromelysin. Although most experimentalfindings suggest that the actions of NO in the cartilage aredetrimental, there is also evidence for protective functions of NO. In arecent study, intravenous inoculation with S. aureus inducedsignificantly increased clinical severity of septic arthritis, withattendant septicemia in iNOS deficient mice, compared with similarlyinfected heterozygous or wild-type mice. This was associated withenhanced production of IFN-_ and TNF-_ in vivo and in vitro, whichindicated a shift towards increased production of Th1-type cytokines.Apart from antimicrobial activity, other beneficial effects of NOinclude stimulation of proteoglycan synthesis during certain conditions,participation in wound healing, and stimulation of collagen production.Furthermore, NO also is reported to promote mucosal integrity. Theisoform nonselective NOS inhibitor L-NAME worsens acute edematous andnecrotizing pancreatitis; whereas, NO donors reduces pancreatic injury.Indeed, there is increasing evidence that iNOS is beneficial, ratherthan detrimental, for resolving intestinal inflammation. Evidence forthe dual roles of inducible NO in modulating gastrointestinal mucosaldefense and injury. Periodontal diseases and inflammatory bowel disease,both chronic inflammatory diseases, are believed to be related, asinduction of periodontal disease results in gut dysbiosis. Inendothelial cells, NOS prevents apoptosis; whereas, it induces apoptosisin smooth muscle cells. The presence of iNOS in atherosclerotic plaquessuggests a role for NO in atherosclerosis but its exact role is stillunknown. One of the primary functions of the inflammatory response is toheal wounded tissue. Healing commences soon after injury, while acuteinflammation in still in full swing. Interestingly, the cytokine mostassociated with wound healing, TGF-_ 1, may be the most potentsuppressor of iNOS. One of the roles of iNOS in wound healing may be tomodulate TGF-_ 1. Caution with the use of iNOS inhibitors is warrantedin settings that require appropriate wound healing.

It is now clear that NO cannot be rigidly catalogued as either ananti-inflammatory or a proinflammatory molecule, but it can beconsidered a true inflammatory mediator. Inducible, high-level NOproduction mediates a number of inflammatory and infectious diseases byacting both as a direct effector and as a regulator of other effectorpathways. Thus, while in a preferred embodiment, the production byPrevotella present in a person's mouth is adjusted to mimic the levelsobserved in healthy Amish individuals, in certain embodiments, e.g.where other factors indicate that excessive, and thus detrimental levelsof NO are being produced, one aspect of the present invention is toaddress the cytotoxic and damaging actions of NO/RNOS withoutinterfering with essential protective functions. Besides selectivelyinhibiting iNOS, a number of other therapeutic strategies areconceivable in order to alleviate the deleterious effects of excessiveNO formation. These alternative therapies involve scavenging of NO/RNOS,and/or inhibition of metabolic pathways triggered by these molecules.The advantage of preserving the beneficial effects of iNOS also needs tobe considered when implementing any therapeutic approach. Theidentification of the roles of NO and of the cells that produce it, aswell as the more complete elucidation of the mechanisms that regulateits cellular production in inflammation, will help in the development oftherapeutic applications for both acute and chronic inflammatorydiseases. Mitochondria preserved some key features of prokaryotesynthesis, demonstrating the evolutionary basis to the NO synthesizingprokaryote world.

In particular embodiments, a mucosal, e.g. oral, strip is provided withan effective amount of an agent effective to non-systemically reducenumbers of undesired bacteria in an individual's oral cavity. In oneembodiment, the agent comprises Azithromycin, which has been found to beeffective against anaerobes and gram-negative bacilli. The provision ofan oral strip enables such drug to be contacted directly with the sitesof inflammation. Still other strips contain an effective amount ofmetronidazole, which targets obligate anaerobes. In preferredembodiments, ciprofloxacin and other similar drugs that targetfacultative anaerobes—Staphylococcus, Corynebacterium, enteric GNRs,etc. are not employed. The use of strips as described herein is a localdelivery method to administer antibiotics and offers a novel approach tothe management of periodontal “localized” infections. One of the primaryadvantages is that smaller doses of topical agents can be deliveredinside the gingival pocket, avoiding the side effects of systemicantibacterial agents, while increasing the exposure of targetmicroorganisms to higher concentrations and therefore more therapeuticlevels of the medication. In still other embodiments, strips areimpregnated with zinc salts as they are non-toxic and do not stain teethcompared with other metal salts. The zinc is believed to assist in theinhibition of growth of undesired bacteria in the oral cavity. Moreover,in other preferred embodiments, strips include explicitly exclude, e.g.are devoid of, either manganese or cooper, as it is known thatspirochetes use the same, instead of iron.

Another approach to antimicrobial therapy in the control of infectionassociated with periodontitis is the concept of full mouth disinfection.In certain embodiments, this is employed in concert with subsequent oralstrip use. The procedure consists of full mouth debridement and thebrushing of the tongue with chlorhexidine gel and then the mouth isrinsed with chlorhexidine solution so that periodontal pockets areirrigated with chlorhexidine solution. Chlorhexidine rinses arepreferred and continued for several weeks to aid healing and augmentplaque control. Systemic administration of doxycycline with full mouthdisinfection is designed to result in better improvement of periodontalparameters and elimination/suppression of putative periodontalpathogens. Prior to or in association with the oral strip, repopulationof the mouth with beneficial bacteria, and preferably those found inhealthy Amish individuals, is believed to be the most effective way tomaintain oral health in a manner that will prevent later Alzheimer'sdisease.

Periodontitis is basically a result of inflammation caused due to widearray of pathogenic microorganism. These microorganisms release numerousproteolytic enzymes, resulting in destruction of soft and hard tissuessupporting the teeth. Protease or peptidase is one of the majorvirulence factors of Prevotella intermedia. Besides its role indegrading the host tissue, proteolysis is also an important part of thesignaling pathway involved in various pathologies including inflammatorydiseases.

Antimicrobials have been used extensively as growth promoters inagricultural animal production, but the specific mechanism of action forthem has not yet been determined. Tylosin administration has been foundto decrease the proportion of bacteria in the phyla Bacteroidetes, ofwhich Prevotella is a member. Despite widespread use of antibiotics forthe treatment of life-threatening infections and for research on therole of commensal microbiota, our understanding of their effects on thehost is still very limited. Several studies have demonstrated thattetracyclines, the antibiotics most intensively used in livestock andthat are also widely applied in biomedical research, interruptmitochondrial proteostasis and physiology in animals ranging from roundworms, fruit flies, and mice to human cell lines.

Prophylactic low-dose antibiotics administered to livestock populationsresult in an increase in the rates of growth and weight gain, promptingtheir widespread use as agents to promote growth in commercial animalherds. Although some of this effect is likely due to changes in the gutmicrobiome, some believe that these effects result from a low-levelincrease in the release of mitochondrial ROS. Low levels ofmitochondrial ROS are essential for cellular proliferation,differentiation, and metabolic adaptation.

In particular embodiments, the use of CRISPR systems to target virulencefactors of bacteria, and in particular, Prevotella is employed to enablethe maintenance and/or destruction of particular microbial populationswhen desired. For example, some of the virulence factors that may betargeted include the following: Gingipain; Capsular polysaccharide;fimbriae; etc. and the employment of CRISPR-Cas systems are used toreduce the virulence factors thereof. While antibiotic therapy isnon-discriminatory in its action, the use of CRISPR systems permits oneto fine tune the selective elimination of particular microbes. Onesurvival mechanism of Prevotella cells is the possession of naturalantibiotic resistant genes, which prevent extermination. Modification ofPrevotella using a CRISPR-Cas system, provides a way to renderPrevotella susceptible to antibiotics, thus permitting its regulation.Certain antibiotics found useful in treating Prevotella includemetronidazole, amoxycillin/clavulanate, ureidopenicilins, carbapenems,cephalosporins, clindamycin, and chloramphenicol.

One aspect of many embodiments of the present invention include the useof specialized viruses to supply CRISPR/Cas to rid bacteria ofantibiotic-resistance plasmids and/or other virulence factors. Virulencefactors of Gram-negative anaerobes such as Prevotella include, forexample, fimbria, hemolysins, adhesions and hemagglutinins. Thesebacteria commonly produce immunoglobulin-degrading enzymes and someproduce tissue-degrading enzymes. Additionally, bacteria of the genusPrevotella are often resistant to antibiotics, such as tetracycline,erythromycin, and .beta.-lactam antibiotics. In practice, aPrevotella-targeting lambda phage is created that encodes the CRISPRgenes plus spacers that target two conserved .beta. lactamases, enzymesthat confer resistance to .beta.-lactam antibiotics. Once integratedinto the Prevotella genome, the phage prevents the transfer of .beta.lactamase-encoding plasmids and can also delete these plasmids fromindividual bacterial cells. These lambda phage-encoding bacteria thenbecome sensitive to treatment with antibiotics.

In one embodiment, an antibacterial rinse is employed, such as thatdescribed in U.S. Pat. No. 8,496,914 to Bonfiglio, and such methodcomprises rinsing with an antibacterial oral rinse formulation for aperiod of time immediately prior to engaging in oral hygiene activities.After effectively killing a majority of Prevotella in a person's mouth,the person reestablishes a population of Prevotella in their mouth byswishing their mouth with a solution containing Prevotella bacteria thathave been modified as described herein. In certain embodiments,virulence factors that are targeted include proteins involved in hostcell attachment and invasion (e.g., fimbriae and adhesins), cytotoxicity(e.g., haemolysins and toxins), iron-acquisition (e.g., siderophores)and evasion or disruption of host-cell defences (e.g., capsule). Genesencoding these factors have been shown to be linked to plasmids and thedistinct chromosomal regions that are termed pathogenicity islands.

In various embodiments of the present invention, bacterial DNA isaltered from pathogenic to non-pathogenic using various methods known tothose of skill in the art. One such method is the employment of aCRISPR-Cas system to introduce a mutation to the bacterial genome, andparticularly to a Prevotella bacterium, including to specific genesencoding for membrane or secretory products, and/or other genes thatregulate virulence genes. Interference with the expression or efficacyof various pathogenic characteristics of certain bacteria, such as byaffecting particular virulence factors possessed by bacteria, viruses,fungi, and protozoa, including but not limited to immunoglobulin (Ig)proteases, capsules, endotoxins, mobile genetic elements, plasmids, andbacteriophages. Specifically, and to provide representative examples,virulence factors for Staphylococcus aureus include hyaluronidase,protease, coagulase, lipases, deoxyribonucleases and enterotoxins. ForPrevotella, proteins involved in adherence to surfaces and/or othercells are modified to reduce competent biofilm formation. Examples forStreptococcus pyogenes are M protein, lipoteichoic acid, hyaluronic acidcapsule, destructive enzymes (including streptokinase, streptodornase,and hyaluronidase), and exotoxins, including streptolysin. Examples forListeria monocytogenes include intemalin A, intemalin B, lysteriolysinO, and actA. Examples for Yersinia pestis include an altered form oflipopolysaccharide, and YopE and YopJ pathogenicity. Other virulencefactors include factors required for biofilm formation (e.g. sortases)and integrins (e.g. beta-1 ad3). In addition to bacteria, helminthespossess similar factors, such as neutrophil inhibitory factor. Thus, oneaspect of the present invention is to employ CRISPR systems to achieveinterference with specific virulence factors or with regulatorymechanisms that control the expression of multiple virulence factors,and in such a manner, provide a way for such microbes to positivelyaffect a person's immune system without attendant pathogenicity. Incertain embodiments this may take the form of employing CRISPR loci tocontrol, for example, the dissemination of antibiotic resistance inbacterial species, such as staphylococci. For example, CRISPR targetingof Streptococcus pneumoniae capsule genes, essential for pneumococcalinfection, provides a way to thwart bacteria virulence and pathogeniceffects. In certain embodiments, the CRISPR-Cas systems are effectivelyemployed as a regulator of gene expression and in such manner, providesa way for bacteria, especially pathogenic bacteria, instead of beingeliminated by the use of broad based antibiotics, are transformed intonon-pathogenic microorganisms, thus maintaining the positive attributesthat they provide in a microbiome of an individual.

FIG. 4 shows close up views of spirochetes, illustrating their spiralnature and conformation. Typical applications and administrationtechniques of antibiotics may not eliminate oral spirochetes as in otherspirochetal diseases, such as syphilis. Typical administration ofantibiotics is believed to only force the spirochetes into a moreprotected spore form, thus triggering a survival strategy that allowsthem to resurface at a later date. One aspect of the present inventionis therefore directed to effective administration of an antibiotic thatwill effectively reduce the population of spirochetal microbes that arecausative of Alzheimer's disease via the direct application ofantibiotics via oral mucosal adhesive strips. Preferably such strips areapplied in an environment where the pH of saliva is adjusted to be below6.5, which is believed to be a level where spirochetal activity is oftenobserved. Most spirochetes are free-living and anaerobic. The stripsthemselves may be adapted to reduce the pH of the oral cavity. While anacidic pH seems to favor certain bacteria, specifically most aerobicbacteria and alkaline pH levels seem to advance anaerobic bacteriagrowth, one aspect of the present invention is to provide a strip thatis able to adjust the pH of an individual's oral cavity to addressparticular situations so as to favor the growth of certain bacteria overothers. Acidosis (an acidic condition of the body) reduces availableoxygen to the cells and can contribute to the increased presence ofcertain bacteria. Certain embodiments of the present invention aretherefore directed to the purposeful infection of a person withspirochetes bacteria that have been treated with a CRISPR-Cas system torender them sensitive to antibiotics or to otherwise make themvulnerable in a fashion such that they are rendered far easier tocontrol or kill.

FIG. 5 illustrates oral biofilm infections of the human body, showing ahuman tooth, gum and the sites of periodontal disease. Recent reportshave documented that infectious agents also occur in atherosclerosis,cardio- and cerebrovascular disorders, diabetes mellitus, chronic lungand inflammatory bowel diseases, and various neurological andneuropsychiatric disorders. As the focus of the present disclosure isadmittedly on Alzheimer's disease, one of skill in the art willnevertheless appreciate that there is a wide over-lap of the fundamentalmechanisms involved in the treatment of Alzheimer's disease as describedherein and that the teachings herein find application in one or more ofthe above listed conditions, and thus, this specification is intended toand should be considered as encompassing the treatment of suchconditions. FIG. 6 illustrates a site of periodontal disease and toareas of the human body believed to be causally affected by inflammationand bacterial infections stemming therefrom.

A paradigm shift in recent years has led to the consideration of theoral cavity as being critically important in maintaining systemichealth. A basic difficulty in destroying certain bacteria present inoral biofilms is that they are not actively dividing, which makes themresistant to attack by a large group of antibiotics and antimicrobialsthat attack the bacteria only during the active parts of theirlifecycle, e.g., cell division. Certain bacteria can also form spores,which are hard, non-permeable protein/polysaccharide shells or coatings.Spores provide additional resistance to eradication efforts bypreventing attack from materials that are harmful to the bacteria. Thus,one aspect of the present invention is to plan attacks on particularbacteria when they are actively dividing, or alternatively, to employbiocides and antimicrobials that are strongly acidic and/or oxidizing,often involving halogen atoms, oxygen atoms, or both. Common examplesinclude hypochlorite solutions (e.g., bleach), phenolics, mineral acids(e.g., HCl), H₂O₂, and the like. Because dosages of such chemicals mustbe allowed to contact the biofilm or spore for extended amounts of timeto be effective, the oral strips as described herein are well suited forthis purpose and various embodiments of the present invention aredirected to strips that contain one or more of the above describedbiocides and antimicrobials.

In certain embodiments, especially those directed to strips adapted toaddress anaerobic bacteria, such strips are provided such that theyfurther generate an environment in the oral cavity that establishes a pHgreater than 7 and less than about 10. The strips thus employed areeffective at interrupting or breaking ionic crosslinks in themacromolecular matrix of a biofilm, which facilitates passage of desiredanti-bacterial agents through the matrix to the bacteria entrainedtherein and/or protected thereby. Surfactants of known types can furtherbe employed to assist in breaking down biofilms. Using the strips asdescribed, one is able to employ lower concentrations than would bepossible in systemic administrations and still achieve the desired breakdown of the bacterial biofilm. While much of the discussion herein isdirected to Prevotella, one of skill in the art will understand thatother bacteria present in the oral cavity can also be targeted for usein combatting periodontal disease, and thus AD. For example, one of themost studied bacteria implicated in periodontal disease is F. nucleatum.It belongs to the Bacteroidaceae family and is a dominant micro-organismwithin the periodonticum. It is a gram-negative anaerobicspecies of thephylum Fusobacteria, numerically dominantin dental plaque biofilms, andimportant in biofilm ecology and human infectious diseases. Dentalplaque is a complex and dynamic microbial community that forms a biofilmon teeth, and harbors more than 400 distinct species in vivo. F.nucleatum is a prominent component quantitatively and is one of thefirst Gram-negative species to become established in plaque biofilms. Itis a central species in physical interactions between Gram-positive andGram-negative species that are important in biofilm colonization, andcontributes to the reducing conditions necessary for the emergence ofoxygen-intolerant anaerobes: it is considered as an intermediatecolonizer bridging the attachment of commensals that colonize the toothand epithelial surface with true pathogens. F. nucleatum is one of asmall number of oral species that is consistently associated with, andincreased in number at, sites of periodontitis, one of the most commoninfections in humans. F. nucleatum has been shown to be a potent inducerof collagenase 3 (MMP-13) production. This suggests that F. nucleatummay be involved in the pathogenesis of periodontal diseases byactivating multiple cell signaling systems that lead to stimulation ofcollagenase 3 expression and increased migration and survival of theinfected epithelial cells. By eliminating immune cells that areimportant for immune defense against oral bacteria, F. nucleatum cancontribute to the recruitment of other pathogenic bacteria andsubsequently to the initiation and the progression of periodontaldisease. Indeed, positive association between F. nucleatum, P.gingivalis and P. intermedia and Bacteroides forsythus in sub-gingivalplaque samples has been reported. Colonization by P. intermedia wasfound to be due to F. nucleatum, since P. intermedia is not detected ina site unless F. nucleatum was also present. It is believed that F.nucleatum recruits and activates local immune cells, resulting in tissuedestruction and the progression of periodontal disease. Instead ofdebating whether F. nucleatum is commensal or pathogenic, one aspect ofthe present invention is to employ CRISPR-Cas systems to modify suchbacteria to advantageously affect various virulence factors of suchbacteria so as to better address access to biofilms where otherbacteria, such as spirochetes, reside, thus providing a way to reducethe incidence of AD. F. nucleatum has periodontopathogenic propertiesbut also appears to be a very sensitive microorganism such that it canbe targeted with respect to its coaggregration properties, thus allowingone of skill in the art to interfere with biolfilm formations involvingthis bacterium and interfering with its abilities to transport andinteract with other periodontopathogenic bacteria.

With a better understanding of how the microbiota interacts with thehost's physiology gained in the last few years, one aspect of variousembodiments of the present invention is to integrate an individual'smicrobiota into a form of personalised healthcare so as to treat anindividual's diseases more efficiently and in a more targeted fashion.With a more complete understanding of the AD disease process, themanipulation of the oral microbiome is focused on to modulate theotherwise normal course of AD disease progression. While Hippocrates mayhave been correct that food is medicine and medicine is food—and thatall disease begins in the gut—the present inventors believe that beforethe food arrives at the gut, it must pass through the oralmicrobiome—and it is in the oral cavity that one can effectively addressmany of the diseases of the modern era, including AD. Saliva iscolourless, odourless and has a relative density of 1.004-1.009 and a pHof 6.6-7.1. Saliva consists of 99% water and the remainder is organicmolecules such as salivary amylase, mucopolysaccharide, mucin andlysozymes, and some inorganic matter such as Na⁺, K⁺, Ca²⁺, Cl⁻ and thethiocyanate ion. Levels of nitric oxide (NO) have been detected insaliva and gingival crevicular fluid collected from patients withgingivitis, aggressive periodontitis and chronic periodontitis—ascompared to healthy controls. Salivary AM and NO levels distinguishedpatients with aggressive periodontitis from other groups. In contrast,patients with chronic periodontitis, aggressive periodontitis andgingivitis showed increased levels of NO in the gingival crevicularfluid, and higher levels of NO were found in patients with periodontitiscompared with those with only gingivitis.

The microbial conversion of nitrate to nitrite in the oral cavity andthe subsequent conversion to nitric oxide is an important factor incombating various diseases. While for many years, the role of nitrate inthe human body has been under debate and usually not in favor ofnitrate, recently there has been a revaluation of this paradigm. Thepresent inventors submit that the bacterial reduction of nitrate tonitrite in the oral cavity is important in the progression of AD andthat there are health benefits to be derived from the oral presence ofnitric oxide. While elevated levels of nitrate and nitrite areassociated with periodontal disease, this elevation is thought to be aresponse of the immune system against infection or stress. Under acidicconditions, nitrite is converted to nitric oxide and acts as anantibacterial agent. P. intermedia lipopolysaccharide can induce iNOSexpression and stimulate the release of NO without additional stimuli.The ability of P. intermedia lipopolysaccharide to promote theproduction of NO is important in the pathogenesis of inflammatoryperiodontal disease, and thus in the progression of AD. The strips ofthe present invention in various embodiments provide a way to provide apotent inhibitor of biofilms that are implicated in periodontal disease,with proper application thereof being effective to provide subgingivalmargins with anti-periodontal pathogen capabilities so as to combatendodontic biofilms.

Other aspects of the present invention are directed to the use ofcommensal bacteria to thwart the growth and development of biofilms thatare believed to be involved in the progression of AD. Hydrogen peroxideproduction by commensal is believed to be a major mechanism ofinhibition and various species of Streptococcus bacteria are employed incertain embodiments of the present invention to inhibit the growth ofpathogens. Trace mineral micronutrients are also imperative for optimumhost health and balanced levels of trace minerals like iron (Fe), zinc(Zn), selenium (Se) and copper (Cu) are essential to prevent progressionof chronic conditions like periodontitis. Their excess as well asdeficiency is detrimental to periodontal health. This is specificallytrue in relation to Fe. Furthermore, some trace elements, e.g. Se, Znand Cu are integral components of antioxidant enzymes and preventreactive oxygen species induced destruction of tissues. Their deficiencycan worsen periodontitis associated with systemic conditions likediabetes mellitus. In various embodiments of the present invention, theemployment of bacteria that is able to generate desired levels ofhydrogen peroxide are used to adjust the population of an individual'soral bacteria populations.

While the focus of the present invention is admittedly directed to AD,other diseases are also related to the health of the oral microbiome.For example, arteriosclerosis and arthritis. In many host tissues,including the endothelial lining of blood vessels, produce hsp60 as theyrespond to certain stressors like high blood pressure. It is postulatedthat an autoimmune mechanism in which the host responds to foreignhsp60, such as bacterial hsp, could be important in the development ofan undesired formation of a lipid-containing material on the endotheliallining of arteries. It has been found that inflamed gingival tissues ofperiodontal patients exhibit a positive antibody response to both thehsp produced by oral bacteria (e.g. Porphyromonas gingivalis) and tohuman hsp60. This reveals that oral bacteria not only play a role inperiodontal disease, but also are involved in diseases related tohumoral immune mechanisms. For example, antibodies against the hsps ofP. gingivalis react with human hsps exposed on the endothelium andproduce cellular damage.

Similarly, the destruction caused by the inflammatory pathway when atransient infection becomes chronic demonstrates that the treatmentsemployed to address a transient infection can literally turn the bodyagainst itself when the inflammation becomes chronic. The role ofchronic inflammation, and in particular periodontitis and itsassociation with many of today's most prevalent diseases, such ascardiovascular disease, Alzheimer's, cancers, diabetes and autoimmunedisorders, is at the heart of the various embodiments of the presentinvention. Coronary Artery Disease remains the number one cause of deathin the world. While traditional risk factors partially account for thedevelopment of Coronary Artery Disease, chronic inflammation plays arole in the development and propagation of this disease. As a furtherexample, Autism spectrum disorder (ASD) affects a significant number ofindividuals worldwide with the prevalence continuing to grow. It isbecoming clear that a large subgroup of individuals with ASD demonstrateabnormalities in mitochondrial function. Given the ties between microbesand mitochondria, the use of particular antibiotics to kill microbesmust also be considered as to the affect such drugs have on mitochondriaand the impacts on various disease states such as ASD.

In various embodiments, an oral strip that achieves at least two of thefollowing is sought to be accomplished: lower pH; provide NO; providealtered (e.g. reduced virulence factors as compared to native)Prevotella, and/or interfere with biofilm formation.

One will appreciate that this Summary of the Invention is not intendedto be all encompassing and that the scope of the invention nor itsvarious embodiments, let alone the most important ones, are necessarilyencompassed by the above description. One of skill in the art willappreciate that the entire disclosure, as well as the incorporatedreferences, pictures, etc. will provide a basis for the scope of thepresent invention as it may be claimed now and in future applications.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE PRESENT INVENTION

In particular embodiments, the invention is directed toward apreventative treatment for AD involving the provision of an enhancedpopulation of Prevotella intermedia bacteria in a person's oral cavity,and more preferably being a population of Prevotella that have beenmodified to make them, one of: more susceptible to antibiotics; alteredin their ability to adhere to surfaces, thus negatively affecting theirabilities to form biofilms. In preferred embodiments, this is achievedby employing the CRISPR-Cas or CRISPR-Cpf1 systems as described herein.The Prevotella bacteria, and in particular Prevotella intermedia, as aknown pathogen and a major cause of bacteria present in inflammatoryperiodontal disease, makes it an odd and remote choice for a positiveand beneficial bacteria by which to combat AD, and thus, the presentinventors believe that this alone provides a teaching away from the useof Prevotella by one of skill in the art. While not bound by theory, itis believed that Prevotella possesses the beneficial capacity to producebeneficial amounts of nitric oxide (NO) and to foster the expression ofinducible nitric oxide synthase (iNOS). Use of especially modifiedPrevotella that reduces biofilm formation is believed to be particularlyadvantageous as a way to reduce the progression of AD.

One aspect of the present invention is directed to the premise thatPrevotella is related to Alzheimer's Disease. Clinical andepidemiological studies reveal that the loss of teeth is associated withpoor memory. The Prevotella bacteria, and in particular, Prevotellaintermedia, is a known pathogen and a major cause of inflammatoryperiodontal disease. To achieve the goal of finding a prophylactictreatment for AD, the puzzle contributing to its pathogenesis must besolved and the relationship between oral infection and the etiology oflate onset AD discerned. The present inventors believe such puzzle hasbeen solved as described herein.

In the cascade of events causing Alzheimer's disease, oralmicroorganisms play a significant role, particularly anaerobic bacteriasuch as Prevotella spp. and Fusobacterium. The recent observation ofmicrobiome-derived small non-coding RNA (sncRNA) and micro RNA (miRNA)translocation and signaling across endothelial barriers between cellsand tissues, indicates that human neurobiology is impacted by theactions of human microbiome-mediated sncRNA and/or miRNA trafficking.The NF-κB signaling pathway for cyto/chemokine release (TNF-315 α, IL-8)produces free radicals, nitric oxide triggers and apoptosis.

In certain embodiments, the present invention is directed to a methodfor preventing Alzheimer's disease in a human subject who has beendiagnosed with periodontitis, involving administering by local gingivalapplication to the subject an effective amount of an antibioticeffective to kill spirochetes bacteria. Preferably, the method includesproviding at least 0.5 mg of a bacterial composition that includesPrevotella bacteria, and preferrably Prevotella bacteria modified to beless adherent to surfaces, thus negatively affecting biofilm formation.as the predominant bacterial genus, such composition administered atleast 48 hours after the step of administering the antibiotic that iseffective to kill spirochetes bacteria. Preferably administration is viaa composition formulated for application directly to a subject's gumsand more preferably by the application of a mucosal adhesive strippreloaded with the antibiotic. In one embodiment the antibioticcomprises methotrexate. In other embodiments, the antibiotic comprisesdoxycycline and methotrexate, with doxycycline provided at a dose ofabout 20 mg, and even more preferably including a doxycyclinepharmaceutical composition that provides steady state blood level ofdoxycycline of a minimum of about 0.1 μg/ml and a maximum of about 1.0μg/ml.

Preferably, one or both of the antibiotic and the composition areprovided in the form of one of a toothpaste, a mouthwash, a chewing gum,a dental floss, a food product, a gel, a slow-release gel, a tablet, agranule, a film, and most preferrably a dissolvable strip. In certainembodiments, a mucosal adhesive strip contains immunotherapeutic agentseffective to treat periodontitis and include a strip impregnated with anagent adapted to locally target anaerobic bacteria at the site of a guminfection and where the agent is provided in an effective amount to killspirochetes bacteria, preferably adapted to treat an oral environmenthaving a pH of lower than about 6.5.

In one embodiment, a mucoadhesive strip containing immunotherapeuticagents is employed to treat periodontitis, even more preferably amucoadhesive strip impregnated with an agent of choice to target locallyanaerobic bacteria at the site of infection.

Various methods involve a predetermination that the subject is sufferinga periodontal disease and is over the age of 40 years old and thecomposition is effective to kill gram negative anaerobic bacteria. Thevarious methods and systems and compositions are configured to permit anindividual to avoid the ravages of Alzheimer's disease and are alldirected to preventing the progression of Alzheimer's disease fromdeveloping. In still other embodiments, the oral bacterial compositionis selected to mimic the oral bacterial composition of healthy Amishindividuals. In one such embodiment, in view of the prevalence of oralPrevotella by Amish individuals as compared to non-Amish persons, theinvention includes the provision of an oral composition containingPrevotella on at least a weekly basis, preferably in the form of alozenge, mouthwash, gum, strip or toothpaste. Such composition ispreferably capable of generating nitric oxide in an amount of sufficientto promote oral microbiome health similar to the microbiome health ofAmish individuals. As part of certain methods, there is a step ofassessing the likelihood that an individual will suffer Alzheimer'sdisease by analyzing the status of such person's oral health in terms ofwhether they suffer from periodontitis. Those persons having a diagnosisof periodontitis are then treated with an antibiotic suitable to killgram negative anaerobic bacteria, specifically an antibiotic regimeneffective in substantially eliminating spirochetes from the individual'soral cavity. Preferably, this is via a strip as described herein.Another aspect of the present invention is to determine if a person'soral health reflects a case of gingivitis rather than periodontitis, andin such instance, not administering antibiotics in particular, butrather, a strip with desired bacteria and/or xylitol. The effectiveadministering of antibiotics to address periodontitis may entail adirect topological application, and preferably does not involve asystemic oral administration of an antibiotic due to the localized andspecialized nature of oral infections and inflammation of the oralcavity associated with dental plaque.

While not bound by theory, but buttressed by experimental and clinicalfindings throughout the years, it is submitted that the vast majority ofAD is caused by the presence of spirochetes traveling form the oralcavity of an individual, to the person's brain. Once there, the body'simmune system reacts by forming amyloid plaques to surround the slowreproducing spirochetes and in the process, causing the havoc thatresults in the symptoms of AD. It is reflective of the serendipity oflife that the causative agent of Alzheimer's Disease has a corkscrewshape. At age 37, after the death of his wife, a formerly rich widow,Alzheimer was financially independent and could indulge in an occasionalbottle of wine. Perhaps in some cosmic comedy, Alzheimer's opening ofhis cherished wines would bring a smile to the Fates contemplating theironies of life. Spirochetes traverse brain matter by corkscrewingthemselves into tissue, only to be eventually enveloped into a stickygoo of amyloid plaque, a spiderman's web designed to stop harm thattragically causes more damage than its original instigator could everhave hoped to inflict. No doubt Alzheimer reminisced about Dr. Gudden,his deceased colleague from their days serving at Frankfurt's MunicipalAsylum for the Mentally III. Gudden was drowned by Crazy King Ludwig IIin an unsuccessful attempt to stop the King's suicide. Alzheimer himselfdied in 1915 at the age of 51—ironically from a chronic bacterialinfection.

It is well known that nitrites are increased in saliva from patientswith periodontal disease. In the oral cavity, nitrites may derive partlyfrom the reduction of nitrates by oral bacteria. Nitrates have beenreported as a defense-related mechanism. The possibility that thesalivary glands respond to oral infectious diseases by increasingnitrate secretion is surmised. Thus, one way to determine whether anindividual is a prospect for suffering from Alzheimer's disease is totest whether the saliva of such person has elevated nitrite levels ascompared to normal individuals, such as a healthy Amish individual.

In other embodiments of the present invention, the step of determiningthe presence of spirochetes in the brain of a subject is made prior tothe application of medications adapted to address periodontitis. Incertain embodiments, an individual at risk of further developingperiodontitis is administered doxycycline plus methotrexate, preferablya low dose (about 20 mg) of doxycycline twice daily, and most preferablyvia a strip as described herein.

One object of the present invention is to provide a mucoadhesive stripthat contains a once-daily pharmaceutical composition containingdoxycycline that will treat adjacent tissue and reduce anaerobicbacteria and especially spirochetes. Such strips may give steady stateblood levels of doxycycline of a minimum of about 0.1 μg/ml and amaximum of about 1.0 μg/ml. Another aspect of the invention is animmediate release strip having a formulation of doxycycline containingless than 50 mg but more than 25 mg, preferably about 40 mg. doxycyclinebase.

One aspect of the present invention is directed to a method and systemthat includes providing a person with a composition, particularly one ormore oral compositions, that include a collection of bacteria,preferably that include the genus Prevotella, that produces nitric oxideto reduce the chances of an individual suffering from Alzheimer'sDisease. In certain embodiments, such method and system involve the oraladministration of a composition comprising Prevotella bacteria capableof producing nitric oxide in a manner such that the person's microfloraand bacterial environment in their oral cavity is populated with suchPrevotella bacteria. Most preferably, the Prevotella is attenuated toeliminate one or more virulence factors, such as adherence abilitiesuseful in forming biofilms.

Certain methods provide for preventing the onset or progression of AD bygene editing, e.g., using CRISPR-Cas9 mediated methods to alter genes inPrevotella bacteria in order to promote the growth of such bacteria suchthat increased amounts of beneficial NO are produced. There appears tobe a relationship between Nitric Oxide and Alzheimer's disease. Lack ofNO production by endothelial cells due to too much ABP aggregateformation and/or free radical generation contributes to the pathology ofAD. It is speculated that if NO production could be assured then ADwould be significantly halted in its track. NO seems to affect theproduction of ABP but does not affect its clearance, thus suggestingthat NO donors may work as preventative of AD, rather than reversing AD.When NO production was increased by use of a known stimulator of NOproduction in an animal model of AD, the levels of ABP decreased andthere was a significant improvement in memory of animals. Many diseasesare characterized by or associated with insufficient nitric oxideproduction. Experimental and clinical studies demonstrate thatinsufficient nitric oxide production is associated with majorcardiovascular risk factors, such as hyperlipidemia, diabetes,hypertension, smoking and atherosclerosis. Nitric oxide production isalso a predictive indicator of future atherosclerotic diseaseprogression. The ability to generate nitric oxide decreases with ageresulting in increased risk of heart and vascular disease. Thus, variousembodiments are directed to providing beneficial bacteria in the oralcavity that produce NO.

Commensal organisms in the oropharyngeal microbiome may be pathogens orpathogenic under certain circumstances. Healthy individuals commonlyharbor low numbers of oral pathogens. The current consensus is thatnormal commensals may become pathogenic when oropharyngeal dysbiosis ispresent. Oropharyngeal dysbiosis may arise when various interrelatedfactors such as diet, salivary flow, pH, immune defenses, and microbialinteractions are not kept in balance. Oropharyngeal microbes “seed” therest of the gastrointestinal tract. It is estimated that 1011 bacterialcells per day flow from the mouth to the stomach. There is a 45% overlapbetween the oropharyngeal and colonic microbiota suggesting that theoral microbiome strongly influences the composition of the gutmicrobiome. Pathogenic biofilms in the mouth, if not treated,potentially provide a continual source of pathogenic microbes to thegut. This may contribute to chronic or recurrent stomach, smallintestine, or colon dysbiosis.

Inflammatory bowel disease and periodontal disease often present ascomorbidities and similar immune pathogenesis have been hypothesized.People with IBD are more likely to have periodontitis than healthysubjects. Patients with IBD and untreated periodontitis have higheropportunistic bacteria populations in inflamed periodontal tissue thanthose with periodontal disease alone.

Other researchers have commented on the prior research of scientists inthe field of AD who have at various times suggested that spirochetes mayplay a causative role in AD. But confoundingly, such researchers failedto truly appreciate what biologic processes were at issue and therefore,failed to appreciate how to treat AD in a fashion so that it could beprevented. Indeed, while some earlier researchers speculated thatspirochetes might play a role in AD, it was thought (incorrectly) thatspirochetes may be the source of the beta amyloid deposited in the ADbrain. This is believed to have led the majority of researchers to focusefforts, time and money on figuring out ways to address amyloid plaquedispersion, rather than address the root cause of AD. In other words,without a clear understanding of the biological and bacterialinterworking of AD, researchers were unable to comprehend in a usefulfashion what some earlier clinical results portended. Even with theassociation between spirochetes infection of a person's brain and thebody's own production of amyloid proteins to address such an infection,those of skill in the art failed to appreciate how best to address thesituation, as the thick biological barriers prevented the use of drugsthat would kill spirochetes. As a result of the confusion reigning inthe field, decades have passed where researchers have sought “cures” forAD that were focused on attempts to break down amyloid features. But bydoing so, however, there is a risk that the invasive spirochetes willthen be released in a fashion that they will cause further damage andharm. In short, even if the current attempts to resolve amyloid buildupin the brain is successful, such an event will not necessarily addressthe true cause of AD—and indeed, may make things worse for individualswhen numerous trapped spirochetes are freed to do further harm.

Still others have simply advocated that the best way in which to treator prevent AD is to live a healthy lifestyle, adopt behavioral andlifestyle habits that provide good health, eat an avocado instead ofpotato chips, reduce sugar intake, drink in moderation and quit smoking.The present inventors consider such advice, as well intentioned as itmay be, to be utterly unhelpful in addressing the root cause of AD. Whatis required is a working understanding of the spirochete infectionpathways involved and how best to reduce the opportunities for oralbacteria to travel to the brain to initiate the AD progression.

AD is an infectious and chronic disease with the involvement of theimmune system in reaction to the establishment of biofilms from oralbacteria that have traveled to the brain from the oral cavity. Suchbacteria then develop physical structures that initiate a parade ofhorribles in terms of how the body's immune system attempts to defeat afoe, all the while destroying the neural tissue surrounding the newbiofilm and amyloid protected spirochetes. The destruction or dissolvingof such biofilms has its own risks, as the achievement of such goal willresult in the freeing of the infecting spirochete that causes the damagein the first place. Moreover, the adaptive immune system rapidlydestroys brain tissue surrounding amyloid plaques, and explains whyafter the 30-odd years that it typically takes for AD to develop, ittakes a mere 3 or so years for considerable damage to be done to aperson's long infected brain. The older brains are different fromyounger brains in that such adaptive immune system responses are notpermitted due to an intact blood brain barrier of younger individuals.Traumatic injuries to the brain tissue, however, permit immune responsesto be observed in the brain. Support for this appreciation can be foundin the NFL player's younger brains that have suffered repeated andviolent trauma due to collisions and concussions. Such hemorrhagiccerebrovascular events like chronic traumatic encephalopathy (CTE)results in the formation of the amyloid structures seen in much older ADvictims.

In terms of treatment, and as otherwise discussed herein, the provisionof antibiotics effective against spirochetes is called for—with thecaveat that they are administered early enough: e.g. prior to theestablishment of protective biofilms such that the spirochetes cannot bekilled. Penicillin administered before the disease starts is believed tobe curative of AD and precludes it from taking its otherwise terriblepath to destroy the mind.

Importantly, and again, not bound by theory, the present inventorsbelieve that the rise in the occurrences of AD in the elderlypopulations has a correlation with dental practices that involve thebleeding of gums, thus presenting occasions where spirochetes can enterthe blood stream and travel to the brain. Some have commented on thefact that while there have been very old individuals living in the UScenturies ago, the number of AD cases has risen far faster than onewould expect. The present inventors submit that the advent of moderndentistry, with wisdom tooth extractions, gum procedures, rough dentalcleanings where blood is often a result, has unwittingly provided a pathfor the spirochetes resident in everyone's mouth to gain access to theblood stream, and thus the brain tissue, of millions of individuals inan unprecedented fashion. The more recent practice of providingantibiotics prior to or at the time of major dental procedures is abeneficial happening, but without a firm understanding of the causativefactors of AD, protective procedures using antibiotics cannot beimplemented in a thoughtful fashion to prevent the spread of AD.

As a practical matter, the infection (or re-infection) of a person'smouth with undesired bacteria is caused by the dental practices ofindividuals. For example, individual's toothbrush heads are typicallycontaminated as most people simply rinse the brush with plain tap water.Unfortunately, it is common for toothbrushes are routinely reused formonths and sometimes for more than a year. The head of a toothbrushcontains up to 100 million germs including many pathogenic bacteria thatcontribute to biofilm formations that lead to AD due to conveyance ofspirochetes to brain tissue via the blood or nervous system pathways.

It is believed that because tooth brushing has become a universallyaccepted first line of defense against illness, and despite goodintentions, the widespread practice of spreading spirochetes in the oralcavity and providing opportunities for them to enter the blood streamvia disruption of the gums, causing bleeding thereof. In other words,while removal of pathogens from the mouth by toothbrush when usedcorrectly has some benefits, it also leaves the toothbrush headcontaminated as most people simply rinse the brush with plain tap water.In addition to bacteria and other microorganisms from the mouth,toothbrushes are often kept near dirty toilets and sinks, furtherincreasing the possibility of contamination. Aerosols released afterflushing the toilet have been shown to deliver fecal matter up to 20feet in the air, reaching toothbrushes that have been stored on bathroomcounters. It has been reported that toothbrushes could be a source ofrepeated oral infection.

Wisdom teeth have only become a problem in the last few centuries. Ourjaws have gotten quite a bit smaller in response to the fact that we eatmore processed and cooked foods than our ancestors did. In those oldertimes when our jaws had begun to shrink, but not by so much as today,the teeth would get stuck, but not cause an infection or any bitingproblem much of the time. Moreover, unlike today—when wisdom teeth areroutinely pulled even prior to wisdom teeth breaking the tissue surface,in olden days such wisdom teeth never did arise, and thus far fewerwisdom teeth extractions were seen. The first dentists did not graduatefrom a modern dental school until about 1870. Thus, the dentalprofession and the opportunities for an individual's gums to experienceperiodic and unprecedented trauma arose in tandem, and long with it, therise in AD appeared from what was once an obscure condition. Alzheimerfirst diagnosed the disease in 1906 and Alzheimer died in 1915 at theage of 51—and had just two patients in his short lifetime that hereported as having the disease he is named for. Contrast this with thefact that Alzheimer's disease is currently ranked as the sixth leadingcause of death in the United States, and recent estimates indicate thatthe disorder may rank third, just behind heart disease and cancer, as aleading cause of death for older people.

As further explained and described herein the provision of oral surfacesthat inhibit the formation of undesired biofilms, whether used inconcert with antibiotics or anti-biofilm agents or not, is anotherbeneficial way in which to prevent the likelihood of AD progression. Oneobjective of the present invention is to treat the conditions thatresult in the decades later development of AD—and such a temporal spanmakes it difficult to appreciate the benefits to be derived fromsensible use of antibacterial structures, agents, etc. But if employed,the teachings of the present invention will assuredly significantlycontribute to the future health of the next generation, such that theoccurrence of AD can be expected to drop, and may eventually fade to itslevels as seen in centuries past.

The present inventors submit that there is a significant associationbetween spirochetes and Alzheimer's Disease. Spirochetes have beenobserved in the brain in more than 90% of Alzheimer's Disease cases.Various species of spirochetes are believed to contribute to AD,including the periodontal pathogens Treponemas (T. pectinovorum, T.amylovorum, T. lecithinolyticum, T. maltophilum, T. medium, T.socranskii). Persisting inflammation and amyloid deposition initiatedand sustained by chronic spirochetal infection results in the tragicprogression of AD. Because spirochetal infection occurs years or decadesbefore the manifestation of dementia, effective protocols for antibioticand anti-inflammatory therapies are required—and are describedherein—such that AD, which like syphilis, is a spirochetes caseddisease—can finally be avoided or at least reduced.

While the present application is primarily directed to AD, one of skillin the art will further appreciate that other disorders that are relatedto inflammatory diseases are also sought to be addressed by employingvarious systems and methods as disclosed herein. For example,atherosclerosis (AS) is a chronic disorder characterized by theformation and progression of plaques within arteries. Various microbes,most notably periodontal organisms, have been identified in plaques bothepidemiologically and microbiologically, and the present inventorscontend that they are contributors to the disease.

While specific embodiments and applications of the present inventionhave been described, it is to be understood that the invention is notlimited to the precise configuration and components disclosed herein.Various modifications, changes, and variations which will be apparent tothose skilled in the art may be made in the arrangement, operation, anddetails of the methods and systems of the present invention disclosedherein without departing from the spirit and scope of the invention.Those skilled in the art will appreciate that the conception upon whichthis disclosure is based, may readily be utilized as a basis fordesigning of other methods and systems for carrying out the severalpurposes of the present invention to instruct and encourage theprevention and treatment of various human diseases. It is important,therefore, that the claims be regarded as including any such equivalentconstruction insofar as they do not depart from the spirit and scope ofthe present invention.

What is claimed is:
 1. A method of reducing the likelihood of aPorphyromonas gingivalis infection in a human being, comprising,providing a bioadhesive strip to a human being who has been diagnosedwith periodontitis, wherein said strip has a first and second side, thefirst side having a bioadhesive that is adapted to bind to a mucosalmembrane for at least 1 hour while inside a person's mouth, said stripbeing devoid of at least one of copper and manganese, and wherein saidstrip includes at least about 200 mg xylitol; administering to the humanbeing Porphyromonas gingivalis that has been modified by using aclustered regularly interspaced short palindromic (CRISPR) CRISPRassociated protein (Cas) system or a CRISPR/Cpf1 system to reduce thevirulence factor gingipain.
 2. The method of claim 1, wherein said stripis devoid of manganese.
 3. The method of claim 1, wherein said stripfurther comprises an effective amount of paquinimod to inhibitcollagenase activity.
 4. The method as set forth in claim 1, whereinsaid strip includes between 0.2 and 0.9% xylitol by weight.
 5. Themethod as set forth in claim 1, wherein said strip comprisesbioluminescent material.
 6. The method as set forth in claim 1, whereinsaid strip has a surface topography for resisting bioadhesion and apattern defined by a plurality of spaced apart features attached to orprojected into a base surface, said plurality of features each having atleast one microscale dimension and having at least one neighboringfeature having a substantially different geometry, wherein an averagespacing between adjacent ones of said features is between 0.5 and 5microns.
 7. The method as set forth in claim 1, further comprisingorally administering to the subgingival tooth area of the human being anantibiotic in an effective amount to kill Porphyromonas gingivalisbacteria residing on the subgingival tooth area of the human beingwherein said antibiotic comprises one of azithromycin and ceftriaxone.8. A method of reducing the likelihood of a Porphyromonas gingivalisinfection in a human being comprising, providing a bioadhesive strip toa human being who has been diagnosed with periodontitis, said stripbinding to a mucosal membrane of said human being, the strip having afirst and second side, the first side having a bioadhesive that binds toa mucosal membrane for at least 1 hour while inside a person's mouth,wherein said strip comprises a therapeutically effective amount of ananti-biofilm agent, and compounds that facilitate the growth of desiredbacteria beneficial to a person's health; and orally administering tothe subgingival tooth area of the human being an antibiotic in aneffective amount to kill Porphyromonas gingivalis bacteria residing onthe subgingival tooth area of the human being, wherein said antibioticcomprises one of azithromycin and ceftriaxone, and wherein the methodreduces the likelihood of a Porphyromonas gingivalis infection in thehuman being and reduces the virulence factor gingipain.
 9. The method asset forth in claim 8, further comprising administering to the humanbeing Porphyromonas gingivalis that has been modified by using aclustered regularly interspaced short palindromic (CRISPR) CRISPRassociated protein (Cas) system or a CRISPR/Cpf1 system to reduce thevirulence factor gingipain.
 10. The method of claim 8, wherein saidstrip is devoid of manganese.
 11. The method of claim 8, wherein saidstrip further comprises an effective amount of paquinimod to inhibitcollagenase activity.
 12. The method as set forth in claim 8, whereinsaid strip includes between 0.2 and 0.9% xylitol by weight.
 13. Themethod as set forth in claim 8, wherein said strip comprisesbioluminescent material.
 14. The method as set forth in claim 8, furthercomprising orally administering to the subgingival tooth area of thehuman being an antibiotic in an effective amount to kill Porphyromonasgingivalis bacteria residing on the subgingival tooth area of the humanbeing wherein said antibiotic comprises one of azithromycin andceftriaxone.
 15. The method as set forth in claim 8, wherein said stripincludes at least about 200 mg xylitol.
 16. The method as set forth inclaim 8, wherein said strip includes an effective amount of paquinimodto inhibit collagenase activity.
 17. The method as set forth in claim 8,wherein said strip has a surface topography for resisting bioadhesionand a pattern defined by a plurality of spaced apart features attachedto or projected into a base surface, said plurality of features eachhaving at least one microscale dimension and having at least oneneighboring feature having a substantially different geometry, whereinan average spacing between adjacent ones of said features is between 0.5and 5 microns.
 18. The method as set forth in claim 8, furthercomprising orally administering to the subgingival tooth area of thehuman being an antibiotic in an effective amount to kill Porphyromonasgingivalis bacteria residing on the subgingival tooth area of the humanbeing wherein said antibiotic comprises one of azithromycin andceftriaxone.
 19. A method of reducing the likelihood of a Porphyromonasgingivalis infection in a human being comprising, providing abioadhesive strip to a human being who has been diagnosed withperiodontitis, said strip binding to a mucosal membrane of said humanbeing, the strip having a first and second side, the first side having abioadhesive that binds to a mucosal membrane for at least 1 hour whileinside a person's mouth, wherein said strip comprises compounds thatfacilitate the growth of desired bacteria beneficial to a person'shealth; orally administering to the subgingival tooth area of the humanbeing an antibiotic in an effective amount to kill Porphyromonasgingivalis bacteria residing on the subgingival tooth area of the humanbeing, wherein said antibiotic comprises one of azithromycin andceftriaxone; and administering to the human being Porphyromonasgingivalis that has been modified by using a clustered regularlyinterspaced short palindromic (CRISPR) CRISPR associated protein (Cas)system or a CRISPR/Cpf1 system to reduce the virulence factor gingipain.20. The method as set forth in claim 19, wherein said strip includes atleast about 200 mg xylitol.